Compositions and systems for conferring disease resistance in plants and methods of use thereof

ABSTRACT

Compositions, systems and methods are provided for conferring disease resistance to plant pathogens that use proteases to target plant substrate proteins inside plant cells. Briefly, the compositions, systems and methods are based upon plant substrate proteins that are targeted by pathogen-specific proteases and that activate nucleotide binding site-leucine rich repeat (NB-LRR) disease resistance proteins when cleaved by the protease. These substrate proteins are modified such that the endogenous protease recognition sequence is replaced by a protease recognition sequence specific to a different pathogen protease (i.e., a heterologous protease recognition sequence). The modified plant substrate protein therefore can be used in connection with its corresponding NB-LRR protein to activate resistance in response to cleavage by the heterologous pathogen-specific protease. When activated by the plant pathogen-specific protease, the pair initiates host defense responses thereto, including programmed cell death.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/700,500, filed on Sep. 13, 2012, which is incorporated byreference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM046451 awardedby the National Institutes of Health. The Government has certain rightsin the invention.

INCORPORATION OF SEQUENCE LISTING

A paper copy of the Sequence Listing and a computer readable form of thesequence containing the file named “31377-21_IURTC13057_ST25.txt”, whichis 17,844 bytes in size (as measured in Microsoft WINDOWS® Explorer),are provided herein and are herein incorporated by reference. ThisSequence Listing consists of SEQ ID NOs:1-28.

BACKGROUND

The present disclosure relates generally to plant genetics and plantmolecular biology, and more particularly relates to compositions,systems and methods of conferring disease resistance to plant pathogensthat express pathogen-specific proteases based on recognition of thepathogen-specific proteases in a plant cell.

Plant diseases are a serious limitation on agricultural productivity andinfluence the development and history of agricultural practices. Avariety of plant pathogens are responsible for plant diseases includingbacteria, fungi, insects, nematodes and viruses.

Incidence of plant diseases can be controlled by agronomic practicesthat include conventional breeding techniques, crop rotation and use ofsynthetic agrochemicals. Conventional breeding methods, however, aretime-consuming and require continuous effort to maintain diseaseresistance as plant pathogens evolve. See, Grover & Gowthaman (2003)Curr. Sci. 84:330-340. Likewise, agrochemicals increase costs to farmersand cause harmful effects on the ecosystem. Because of such concerns,regulators have banned or limited the use of some of the most harmfulagrochemicals.

Agricultural scientists now can enhance plant pathogen resistance bygenetically engineering plants to express anti-pathogen polypeptides.For example, potatoes and tobacco plants have been developed thatexhibit an increased resistance to foliar and soil-borne fungalpathogens. See, Lorito et al. (1998) Proc. Natl. Acad. Sci. USA95:7860-7865. In addition, transgenic barley has been developed thatexhibit an increased resistance to fungal pathogens. See, Horvath et al.(2003) Proc. Natl. Acad. Sci. USA 100:364-369. Moreover, transgenic cornand cotton plants have been developed to produce Cry endotoxins. See,e.g., Aronson (2002) Cell Mol. Life Sci. 59:417-425; and Schnepf et al.(1998) Microbiol. Mol. Biol. Rev. 62:775-806. Other crops, includingpotatoes, have been genetically engineered to contain similarendotoxins. See, Hussein et al. (2006) J. Chem. Ecol. 32:1-8; Kalushkov& Nedved (2005) J. Appl. Entomol. 129:401-406 and Dangl et al. (2013)Science 341: 746-751.

In light of the significant impact of plant pathogens on the yield andquality of plants, additional compositions, systems and methods areneeded for protecting plants from plant pathogens.

BRIEF SUMMARY

Compositions, systems and methods are provided for conferring diseaseresistance to plant pathogens that express pathogen-specific proteasesby modifying at least one member of a protein pair used by plants todetect the pathogen-specific proteases. These protein pairs enableplants to activate endogenous defense systems in response to thepathogen-specific proteases. Briefly, the compositions, systems andmethods are based upon a protein pair in which one member of the pair isa nucleotide binding-leucine rich repeat (NB-LRR) disease resistanceprotein and the other member of the pair is a substrate protein of apathogen-specific protease that physically associates with itsnative/corresponding NB-LRR protein and that activates the NB-LRRprotein when cleaved by the pathogen-specific protease. The specificityof such pairs for a given pathogen-specific protease can be engineeredby replacing an endogenous protease recognition sequence in thesubstrate protein with a recognition sequence for a pathogen-specificprotease of interest (i.e., a heterologous protease recognitionsequence).

The compositions include recombinant nucleic acid molecules having anucleotide sequence that encodes a modified substrate protein of apathogen-specific protease, where the modified substrate protein has aheterologous protease recognition sequence. The heterologous proteaserecognition sequence can be within, for example, an exposed loop of themodified substrate protein. Optionally, the recombinant nucleic acidmolecule can have a nucleotide sequence that encodes a NB-LRR protein sothat the nucleic acid molecule encodes the protein pair. For example, inone embodiment, a recombinant nucleic acid molecule having a nucleotidesequence that encodes the NB-LRR protein can be co-transformed with therecombinant nucleic acid molecules having a nucleotide sequence thatencodes a modified substrate protein of a pathogen-specific protease sothat the modified substrate protein and the NB-LRR protein areco-expressed. The NB-LRR protein can associate with, and can beactivated by, the modified substrate protein of the pathogen-specificprotease.

The compositions also include isolated, modified substrate proteins ofpathogen-specific proteases as described herein, as well as activefragments and variants thereof.

The compositions also include nucleic acid constructs, such asexpression cassettes and vectors, having a nucleotide sequence thatencodes a modified substrate protein of a pathogen-specific protease asdescribed herein operably linked to a promoter that drives expression ina plant cell, plant part or plant. Such a nucleic acid construct can beused to provide a modified substrate protein to a plant cell, plant partor plant that natively expresses the corresponding NB-LRR protein. Themodified substrate protein can associate with, and can activate, theNB-LRR protein.

Optionally, the constructs, including expression cassettes and vectors,can include a nucleotide sequence that encodes a NB-LRR protein operablylinked to a promoter that drives expression in a plant cell, plant partor plant. The nucleic acid constructs having a nucleotide sequence thatencodes a modified substrate protein of a pathogen-specific protease andthe nucleic acid constructs having a nucleotide sequence that encodes aNB-LRR protein can be co-expressed in a plant cell, plant part or plant.The NB-LRR protein can associate with, and can be activated by, themodified substrate protein of the pathogen-specific protease. Such anucleic acid construct can be used to provide the protein pair to aplant cell, plant part or plant that does not natively express bothmembers of the protein pair.

The compositions also include transformed plant cells, plant parts andplants having a nucleotide sequence that encodes at least one modifiedsubstrate protein of a pathogen-specific protease as described hereinoperably linked to a promoter that drives expression in a plant cell,plant part or plant. Optionally, the plant cells, plant parts and plantsare transformed to include a nucleotide sequence that encodes a NB-LRRprotein operably linked to a promoter that drives expression in theplant cell, plant part or plant. The NB-LRR protein can associate with,and can be activated by, the modified substrate protein of thepathogen-specific protease.

The systems include a nucleic acid construct having a nucleotidesequence for a first promoter that drives expression in a plant cell,plant part or plant operably linked to a nucleotide sequence thatencodes a modified substrate protein of a pathogen-specific protease asdescribed herein and a nucleotide sequence for a second promoter thatdrives expression in a plant cell, plant part or plant operably linkedto a nucleotide sequence that encodes a NB-LRR protein. The NB-LRRprotein can associate with, and can be activated by, the modifiedsubstrate protein. Such systems can be used to provide the protein pairto a plant cell, plant part or plant that does not natively express bothmembers of the protein pair.

The systems also include a first nucleic acid construct havingnucleotide sequence for a promoter that drives expression in a plantcell, plant part or plant operably linked to a nucleotide sequence thatencodes a modified substrate protein of a pathogen-specific protease asdescribed herein, and a second nucleic acid construct having anucleotide sequence for a promoter that drives expression in a plantcell, plant part or plant operably linked to a nucleotide sequence thatencodes a NB-LRR protein. Additional nucleic acid constructs also can beincluded in the system, where each construct has a nucleotide sequencethat encodes a distinct modified substrate protein, each having aheterologous recognition sequence for a separate pathogen-specificprotease. Although each modified substrate protein has a heterologousrecognition sequence distinct from one another, each can associate with,and can activate, the NB-LRR protein. Alternatively, the first nucleicacid construct can encode more than one modified substrate protein,where each modified substrate protein has a heterologous recognitionsequence distinct from one another and where each can associate with,and can activate, the NB-LRR protein. Alternatively, the second nucleicacid construct can encode one or more modified substrate proteins, whereeach modified substrate protein has a heterologous recognition sequencedistinct from one another and where each can associate with, and canactivate, the NB-LRR protein. Such systems can be used to provide theprotein pair to a plant cell, plant part or plant that does not nativelyexpress the protein pair or can be used to provide more than onemodified substrate protein to a plant cell, plant part or plant.

By way of example, the substrate protein of the pathogen-specificprotease can be a PBS1 or a RIN4 homolog from Arabidopsis thaliana, andthe NB-LRR protein can be a RPS5 or a RPS2 from A. thaliana, where thePBS1 or RIN4 homolog is modified to include a heterologous proteaserecognition sequence. As understood by those skilled in the art, “PBS1”refers to avrPphB susceptible 1. As understood by those skilled in theart, “RIN4” refers to Resistance To Pseudomonas syringae pv. maculicola1 (“RPM1”) Interacting Protein 4. As understood by those skilled in theart, “avrRpt2” refers to the bacterial avirulence gene from Pseudomonassyringae that encodes the “AvrRpt2” polypeptide having a role inplant-P. syringae interactions (see, Innes et al. (1993) J. Bacteriol175:4859-4869). As understood by those skilled in the art, “avrPphB”refers to the bacterial avirulence gene from P. syringae that encodesthe “AvrPphB” polypeptide having a role in plant-P. syringaeinteractions.

In view of the foregoing, the methods include introducing into a plantcell, plant part or plant at least one nucleic acid molecule, construct,expression cassette or vector as described herein to confer diseaseresistance to plant pathogens that express pathogen-specific proteases.

The compositions, systems and methods therefore find use in conferringdisease resistance to plant pathogens by transferring to plant cells,plant parts or plants nucleotide sequences that encode at least onemodified substrate protein of a pathogen-specific protease andoptionally that encode a NB-LRR protein when such NB-LRR protein is notnative to the plant cell, plant part or plant. The pair is thusengineered to be specific for a plant pathogen-specific protease byincluding in the modified substrate protein a heterologous proteaserecognition sequence for that plant pathogen-specific protease. Whenactivated by the plant pathogen-specific protease, the pair initiateshost defense responses thereto, including programmed cell death.

These and other features, objects and advantages of the presentdisclosure will become better understood from the description thatfollows. In the description, reference is made to the accompanyingdrawings, which form a part hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The features, objects and advantages other than those set forth abovewill become more readily apparent when consideration is given to thedetailed description below. Such detailed description makes reference tothe following drawings, wherein:

FIG. 1 is a photograph showing that RPS5 (a NB-LRR disease resistanceprotein) can be activated by AvrRpt2 (a plant pathogen-specificprotease) when co-expressed with a modified PBS1 (a substrate proteinfor a plant pathogen-specific protease) containing an AvrRpt2 cleavagesite. Indicated nucleic acid molecules were transiently expressed inNicotiana glutinosa leaves by injecting Agrobacterium tumefaciensstrains carrying the indicated nucleic acid molecules. Each nucleic acidmolecule was under control of a dexamethasone-inducible promoter, andleaves were photographed about 24 hours after dexamethasone application.Visible leaf collapse indicates activation of programmed cell death.

FIG. 2 is a graph illustrating electrolyte leakage from N. glutinosaleaf disks inoculated with the same strains as used in FIG. 1. Increasesin electrolyte leakage indicate loss of plasma membrane integrity due tocell death. PBS1^(RCS2) indicates PBS1 in which its AvrPphB (a plantpathogen-specific protease) cleavage site (GDKSHVS; SEQ ID NO:1) wasreplaced with the AvrRpt2 cleavage site (VPKFGDW; SEQ ID NO:2).

FIG. 3 shows photographs of infected leaves from transgenic A. thalianaexpressing PBS1^(RCS2) (i.e., PBS1 in which the AvrPphB cleavage sitewas replaced with the AvrRpt2 cleavage site). Shown are leaves from fivedifferent primary transformants inoculated on the right side withPseudomonas syringae strain DC3000(AvrRpt2). The photographs were taken24 hours after inoculation, a time point at which untransformed A.thaliana leaves do not display cell death. The A. thaliana accessionused for this experiment contained mutations in RIN4 and RPS2, whichprevent activation of cell death by AvrRpt2 in the absence of modifiedPBS1.

FIG. 4 is a schematic representation of a PBS1^(RCS2) constructillustrating the replacement of the AvrPphB cleavage site within thePBS1 activation loop with the RIN4 cleavage site 2 (RCS2) as discussedin Example 5.

FIG. 5 is a photograph taken 24 hours post-induction showing that theco-expression of PBS1^(RCS2) with AvrRpt2 and PRS5 induced anRPS5-dependent cell death response, whereas cell death was not detectedin the absence of AvrRpt2 or PBS1^(RCS2) as discussed in Example 5.

FIG. 6 is a graph illustrating that PBS1^(RCS2) with AvrRpt2 induced asmuch electrolyte leakage as wild-type PBS1 cleaved by AvrPphB, whereasPBS1^(RCS2) with C122A only weakly activated RPS5 as discussed inExample 5. Data represents the mean and standard deviation (n=4).

FIG. 7 is an immunoblot confirming that AvrRpt2 cleaved PBS1^(RCS2) at 6hours post-induction, whereas C122A or AvrPphB did not as discussed inExample 5.

FIG. 8 is a photograph showing the induction of HR in transgenic lines 2and 5 in response to inoculation with P. syringae, whereas lines 1 and 3did not as discussed in Example 5. “Col” indicates the wild-typeArabidopsis parent.

FIG. 9 is a graph illustrating that PBS1^(RCS2) confers resistance tobacterial growth of DC3000(avrRpt2) in Arabidopsis as discussed inExample 5. Data represent mean cfu cm⁻² (n=4), and error bars denotestandard deviation.

FIG. 10 contains immunoblots showing that resistance to bacterial growthof DC3000(avrRpt2) correlated with expression of PBS1^(RCS2) asdiscussed in Example 5.

FIG. 11 is an immunoblot showing cleavage of PBS1^(RCS2) expressed intransgenic Arabidopsis by AvrRpt2 delivered by DC3000 as discussed inExample 5. The asterisk indicates the expected size of the C-terminalPBS1^(RCS2) cleavage product.

FIG. 12 is a photograph showing that PBS1^(RCS2) transgenic Arabidopsisalso displayed HR 21 hours after injection with DC3000(avrPphB),demonstrating that native recognition specificity of RPS5 was retainedin these transgenic lines as discussed in Example 5.

FIG. 13 is a representation of a PBS1^(TCS) construct illustrating thereplacement of the seven amino acids flanking the AvrPphB cleavage sitewith a TEV cleavage site as discussed in Example 5.

FIG. 14 contains immunoblots showing cleavage of PBS1^(TCS)-HA asdetected by anti-HA (upper panel) as discussed in Example 5.

FIG. 15 is a photograph of N. benthamiana co-expressing PBS1^(TCS) withTEV protease activated RPS5 as discussed in Example 5. The left side ofeach leaf was infiltrated with Agrobacterium strains that deliveredPBS1^(TCS), TEV protease and RPS5.

FIG. 16 is a graph illustrating that PBS1^(TCS) with TEV proteaseinduced RPS5-mediated cell death as indicated by electrolyte leakage.The level of electrolyte leakage was similar to that induced bywild-type PBS1 cleaved by AvrPphB as discussed in Example 5. Datarepresents the mean and standard deviation (n=4).

FIG. 17 is a photograph showing AvrPphB recognition by soybean asdiscussed in Example 6.

While the present disclosure is susceptible to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail. It should beunderstood, however, that the description of exemplary embodiments isnot intended to limit the disclosure to the particular forms disclosed,but on the contrary, the intention is to cover all modifications,equivalents and alternatives falling within the scope of the disclosureas defined by the embodiments above and the claims below. Referenceshould therefore be made to the embodiments above and claims below forinterpreting the scope of the present disclosure.

DETAILED DESCRIPTION

The compositions, systems and methods now will be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all embodiments of the present disclosure are shown. Indeed, thepresent disclosure may be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will satisfyapplicable legal requirements.

Likewise, many modifications and other embodiments of the compositions,systems and methods described herein will come to mind to one of skillin the art to which the invention pertains having the benefit of theteachings presented in the foregoing descriptions and the associateddrawings. Therefore, it is to be understood that the present disclosureis not to be limited to the specific embodiments disclosed and thatmodifications and other embodiments are intended to be included withinthe scope of the appended claims. Although specific terms are employedherein, they are used in a generic and descriptive sense only and notfor purposes of limitation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of skill in the artto which the invention pertains. Although any methods and materialssimilar to or equivalent to those described herein can be used in thepractice or testing of the present disclosure, the preferred methods andmaterials are described herein.

Moreover, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one element is present,unless the context clearly requires that there be one and only oneelement. The indefinite article “a” or “an” thus usually includes “atleast one.”

Many plant pathogens employ proteases as virulence factors, includingbacteria, fungi and viruses. As used herein, “plant pathogen” or“pathogen” means an organism that interferes with or is harmful to plantdevelopment and/or growth. Examples of plant pathogens include, but arenot limited to, bacteria (e.g., Xanthomonas spp. and Pseudomonas spp.),fungi (e.g., members in the phylum Ascomycetes or Basidiomycetes, andfungal-like organisms including Oomycetes such as Pythium spp. andPhytophthora spp.), insects, nematodes (e.g., soil-transmitted nematodesincluding Clonorchis spp., Fasciola spp., Heterodera spp., Globoderaspp., Opisthorchis spp. and Paragonimus spp.), protozoans (e.g.,Phytomonas spp.), and viruses (e.g., Comovirus spp., Cucumovirus spp.,Cytorhabdovirus spp., Luteovirus spp., Nepovirus spp., Potyvirus spp.,Tobamovirus spp., Tombusvirus spp. and Tospovirus spp.)

Plants, however, contain innate disease resistance against a majority ofplant pathogens. Natural variation for resistance to plant pathogens hasbeen identified by plant breeders and pathologists and can be bred intomany plants. These natural disease resistance genes provide high levelsof resistance (or immunity) to plant pathogens and represent aneconomical and environmentally friendly form of plant protection.

Innate disease resistance in plants to plant pathogens typically isgoverned by the presence of dominant or semidominant resistance (R)genes in the plant and dominant avirulence (avr) genes in the pathogen.The largest group of R genes encodes proteins characterized by thepresence of a NB-LRR. This form of innate disease resistance typicallyinitiates programmed cell death in infected plant cells/tissues.

A. thaliana, for example, uses R genes to confer resistance to P.syringae strains that express the avr gene, avrPphB. Specificrecognition of AvrPphB requires at least two genes, RPS5 and PBS1. RPS5encodes a NB-LRR disease resistance protein, and PBS1 encodes aserine/threonine protein kinase.

The work described herein is the first to show that an endogenous P.syringae AvrPphB protease recognition sequence within the activationloop of PBS1 (an exposed loop on the surface of the PBS1 protein) can bereplaced with a heterologous protease recognition sequence. Inparticular, it is shown that the endogenous AvrPphB cleavage site(GDKSHVS; SEQ ID NO:1) of PBS1 can be replaced with a heterologousAvrRpt2 cleavage site (VPKFGDW; SEQ ID NO:2) from the Arabidopsis RPM1Interacting Protein 4 (RIN4), thereby producing a modified PBS1 (SEQ IDNO:6) that can be used in connection with RPS5 to confer resistance topathogens that express AvrRpt2 instead of AvrPphB. It also is shown thatthe endogenous AvrPphB cleavage site (GDKSHVS; SEQ ID NO:1) of PBS1 canbe replaced with a heterologous TEV protease cleavage site (VPKFGDW; SEQID NO:4) of a TEV polyprotein, thereby producing another modified PBS1(SEQ ID NO:8) that can be used in connection with RPS5 to conferresistance to pathogens that express TEV protease instead of AvrPphB. Itis further contemplated that that an endogenous P. syringae AvrRpt2cleavage site (VPKFGDW; SEQ ID NO:2) of RIN4 can be replaced with acleavage site of other pathogen-specific proteases, leading to theactivation of its corresponding NB-LRR protein, RPS2, in the presence ofsuch pathogen-specific proteases. It is also contemplated that a SoybeanMosaic Virus cleavage recognition site (SMV Nla protease; EPVSTQG; SEQID NO:27) can replace the AvrPphB cleavage site, thereby producinganother modified PBS1. It is further contemplated that a Bean Pod MottleVirus cleavage recognition site (BPMV NIa protease; PVVQAQS; SEQ IDNO:28) can replace the AvrPphB cleavage site, thereby producing anothermodified PBS1.

The present disclosure therefore provides compositions, systems andmethods for conferring additional disease resistance to plant pathogensthat express specific proteases in plant cells, plant parts or plants byusing a modified substrate of a pathogen-specific protease that has aheterologous protease recognition sequence in connection with itscorresponding NB-LRR protein.

Compositions Recombinant Nucleic and Amino Acid Molecules

Compositions of the present disclosure include recombinant nucleic andamino acid sequences for modified substrate proteins ofpathogen-specific proteases in which an endogenous protease recognitionsequence within the substrates are replaced with a heterologous proteaserecognition sequence.

In one aspect, the present disclosure is directed to a recombinantnucleic acid molecule comprising a nucleotide sequence that encodes atleast one substrate protein of a plant pathogen-specific protease havinga heterologous pathogen-specific protease recognition sequence withinthe substrate protein. The substrate protein can be, for example,AvrPphB susceptible 1 (PBS1) and Resistance To Pseudomonas syringae pv.maculicola 1 (RPM1) Interacting Protein 4 (RIN4). Particularly suitablesubstrate proteins can be, for example, Arabidopsis thaliana AvrPphBsusceptible 1 (PBS1) and Arabidopsis thaliana Resistance To Pseudomonassyringae pv. maculicola 1 (RPM1) Interacting Protein 4 (RIN4).

As used herein, a “nucleic acid” sequence means a DNA or RNA sequence.The term encompasses sequences that include any of the known baseanalogues of DNA and RNA such as, but not limited to 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, -uracil-5-oxyacetic acid methylester, uracil-5-oxyaceticacid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

As used herein, “recombinant,” when used in connection with a nucleicacid molecule, means a molecule that has been created or modifiedthrough deliberate human intervention such as by genetic engineering.For example, a recombinant nucleic acid molecule is one having anucleotide sequence that has been modified to include an artificialnucleotide sequence or to include some other nucleotide sequence that isnot present within its native (non-recombinant) form.

Further, a recombinant nucleic acid molecule has a structure that is notidentical to that of any naturally occurring nucleic acid molecule or tothat of any fragment of a naturally occurring genomic nucleic acidmolecule spanning more than one gene. A recombinant nucleic acidmolecule also includes, without limitation, (a) a nucleic acid moleculehaving a sequence of a naturally occurring genomic or extrachromosomalnucleic acid molecule, but which is not flanked by the coding sequencesthat flank the sequence in its natural position; (b) a nucleic acidmolecule incorporated into a construct, expression cassette or vector,or into a host cell's genome such that the resulting polynucleotide isnot identical to any naturally occurring vector or genomic DNA; (c) aseparate nucleic acid molecule such as a cDNA, a genomic fragment, afragment produced by polymerase chain reaction (PCR) or a restrictionfragment; and (d) a recombinant nucleic acid molecule having anucleotide sequence that is part of a hybrid gene (i.e., a gene encodinga fusion protein). As such, a recombinant nucleic acid molecule can bemodified (chemically or enzymatically) or unmodified DNA or RNA, whetherfully or partially single-stranded or double-stranded or eventriple-stranded.

A nucleic acid molecule (or its complement) that can hybridize to any ofthe uninterrupted nucleotide sequences described herein, under eitherhighly stringent or moderately stringent hybridization conditions, alsois within the scope of the present disclosure.

As used herein, “stringent conditions” means conditions under which onenucleic acid molecule will hybridize to its target to a detectablygreater degree than to other sequences (e.g., at least two-fold overbackground). Stringent conditions can be sequence-dependent and will bedifferent in different circumstances. By controlling the stringency ofthe hybridization and/or washing conditions, target sequences that are100% complementary to the nucleic acid molecule can be identified (i.e.,homologous probing). Alternatively, the stringent condition can beadjusted to allow some mismatching in sequences so that lower degrees ofsimilarity are detected (i.e., heterologous probing).

Typically, stringent conditions can be one in which the saltconcentration is less than about 1.5 M Na⁺, typically about 0.01 M to1.0 M Na⁺ (or other salts) at about pH 7.0 to 8.3, and a temperature ofat least about 30° C. for short molecules (e.g., 10 to 50 nucleotides)and of at least about 60° C. for long molecules (e.g., greater than 50nucleotides). Stringent conditions also can be achieved by addingdestabilizing agents such as formamide.

As used herein, “about” means within a statistically meaningful range ofa value or values such as a stated concentration, length, molecularweight, pH, sequence identity, time frame, temperature or volume. Such avalue or range can be within an order of magnitude, typically within20%, more typically within 10%, and even more typically within 5% of agiven value or range. The allowable variation encompassed by “about”will depend upon the particular system under study, and can be readilyappreciated by one of skill in the art.

An exemplary low stringent condition includes hybridizing with a buffersolution of about 30% to about 35% formamide, 1 M NaCl, 1% SDS (sodiumdodecyl sulphate) at about 37° C., and washing in about 1× to 2×SSC(20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at about 50° C. to about 55°C. Wash buffers optionally can comprise about 0.1% to about 1% SDS.

An exemplary moderate stringent condition includes hybridizing in about40% to about 45% formamide, 1.0 M NaCl, 1% SDS at about 37° C., andwashing in about 0.5× to 1×SSC at about 55° C. to about 60° C. Washbuffers optionally can comprise about 0.1% to about 1% SDS.

An exemplary high stringent condition includes hybridizing in about 50%formamide, 1 M NaCl, 1% SDS at about 37° C., and washing in about0.1×SSC at about 60° C. to about 65° C. Wash buffers optionally cancomprise about 0.1% to about 1% SDS.

The duration of hybridizing generally can be less than about 24 hours,usually about 4 hours to about 12 hours. The duration of the washing canbe at least a length of time sufficient to reach equilibrium. Additionalguidance regarding such conditions is readily available in the art, forexample, in Molecular Cloning: A Laboratory Manual, 3rd ed. (Sambrook &Russell eds., Cold Spring Harbor Press 2001); and Current Protocols inMolecular Biology (Ausubel et al. eds., John Wiley & Sons 1995).

The heterologous pathogen-specific protease recognition sequence can befrom about 5 amino acids to about 15 amino acids. A list of plantpathogen-specific proteases, plant pathogens of origin, proteasesubstrate proteins and the endogenous protease recognition sequences arelisted in Table 1, which can be used as a source for heterologousprotease recognition sequences.

TABLE 1Plant Pathogen-Specific Proteases, Plant Pathogen of Origin, ProteaseSubstrate Proteins and Endogenous Protease Recognition Sequence.Protease Substrate Protein Plant Pathogen- and Plant Pathogen ofProtease Recognition Sequence Specific Protease Origin(amino acid sequence) AvrPphB P. syringae PBS1; GDKSHVS (SEQ ID NO: 1)AvrRpt2 P. syringae RIN4; VPKFGDW (SEQ ID NO: 2) HopN1 P. syringaePsbQ; QEHGCQL (SEQ ID NO: 3) TEV protease Tobacco Etch VirusTEV polyprotein; ENLYFQG (SEQ ID NO: 4) SMV Nla proteaseSoybean Mosaic Virus SMV polyprotein; EPVSTQG (SEQ ID NO: 27)BPMV Nla protease Bean Pod Mottle VirusBPMV polyprotein; PVVQAQS (SEQ ID NO: 28)

Additional avirulence and disease resistance pairs can be found in, forexample, Jones et al. (1994) Science 266:789-793; Martin et al. (1993)Science 262:1432-1436; and Mindrinos et al. (1994) Cell 78:1089-1099).

The examples below relate to the RPS5 substrate protein and PBS1 NB-LRRprotein pair of A. thaliana. Nucleic and amino acids sequences for RPS5are known and characterized. See, e.g., GenBank® Accession Nos.NM_001198041.1, NM_101094.2 and 064973.2. See also, Warren et al. (1998)Plant Cell 10:1439-1452; and DeYoung et al. (2012) Cell. Microbiol.14:1071-1084. Likewise, nucleic and amino acids sequences for PBS1 areknown and characterized, see, e.g., GenBank® Accession Nos. NM_121319.4,NM_115403.3, AF314176.1, NP_196820 and AAG38109.1. See also, Swiderski &Innes (2001) Plant J. 26:101-112; and DeYoung et al. (2012), supra, aswell as U.S. Pat. No. 5,648,599. The pathogen-specific protease nativelyrelated to this pair is AvrPphB (GenBank® Accession No. CAI36057.1).

Other examples relate to the RIN4 substrate protein and RPS2 NB-LRRprotein pair of A. thaliana. Nucleic and amino acid sequences for RIN4and RPS2 are known and characterized. See, e.g., GenBank® Accession Nos.Q8GYN5.1 and AAA21874.1. The pathogen-specific protease natively relatedto this pair is AvrRpt2 (GenBank® Accession No. Q6LAD6.1).

An example of a recombinant nucleic acid molecule encoding a modifiedsubstrate protein of a pathogen-specific protease therefore includes anucleotide sequence that encodes PBS1 in which its endogenous AvrPphBcleavage site (SEQ ID NO:1) is replaced with a heterologous AvrRpt2cleavage site (SEQ ID NO:2), as is shown in SEQ ID NO:5. Another exampleof a recombinant nucleic acid molecule encoding a modified substrateprotein of a pathogen-specific protease includes a nucleotide sequencethat encodes PBS1 in which its endogenous AvrPphB cleavage site (SEQ IDNO:1) is replaced with a heterologous TEV protease cleavage site (SEQ IDNO:4), as is shown in SEQ ID NO:7. Another example of a recombinantnucleic acid molecule encoding a modified substrate protein of apathogen-specific protease includes a nucleotide sequence that encodesPBS1 in which its endogenous AvrPphB cleavage site (SEQ ID NO:1) isreplaced with a heterologous HopN1 cleavage site (SEQ ID NO:3). Anotherexample of a recombinant nucleic acid molecule encoding a modifiedsubstrate protein of a pathogen-specific protease includes a nucleotidesequence that encodes RIN4 in which its endogenous AvrRpt2 cleavage site(SEQ ID NO:2) is replaced with a heterologous AvrPphB cleavage site (SEQID NO:1). Another example of a recombinant nucleic acid moleculeencoding a modified substrate protein of a pathogen-specific proteaseincludes a nucleotide sequence that encodes RIN4 in which its endogenousAvrRpt2 cleavage site (SEQ ID NO:2) is replaced with a heterologous TEVprotease cleavage site (SEQ ID NO:4). Another example of a recombinantnucleic acid molecule encoding a modified substrate protein of apathogen-specific protease includes a nucleotide sequence that encodesRIN4 in which its endogenous AvrRpt2 cleavage site (SEQ ID N0:2) isreplaced with a heterologous HopN1 cleavage site (SEQ ID NO:3). Anotherexample of a recombinant nucleic acid molecule encoding a modifiedsubstrate protein of a pathogen-specific protease includes a nucleotidesequence that encodes PBS1 in which its endogenous AvrPphB cleavage site(SEQ ID NO:1) is replaced with a heterologous SMV cleavage site (SEQ IDNO:27). Another example of a recombinant nucleic acid molecule encodinga modified substrate protein of a pathogen-specific protease includes anucleotide sequence that encodes PBS1 in which its endogenous AvrPphBcleavage site (SEQ ID NO:1) is replaced with a heterologous BPMVcleavage site (SEQ ID NO:28). Another example of a recombinant nucleicacid molecule encoding a modified substrate protein of apathogen-specific protease includes a nucleotide sequence that encodesRIN4 in which its endogenous AvrRpt2 cleavage site (SEQ ID NO:2) isreplaced with a heterologous SMV cleavage site (SEQ ID NO:27). Anotherexample of a recombinant nucleic acid molecule encoding a modifiedsubstrate protein of a pathogen-specific protease includes a nucleotidesequence that encodes RIN4 in which its endogenous AvrRpt2 cleavage site(SEQ ID NO:2) is replaced with a heterologous BPMV cleavage site (SEQ IDNO:28). The endogenous protease cleavage sequence, which is a preferredlocation for the heterologous protease recognition sequence, can belocated in an exposed loop of the substrate protein, for example. In oneparticularly suitable embodiment of the substrate protein, theendogenous protease cleavage sequence can be located, for example,between about amino acid position 240 to about amino acid position 250when the substrate protein is PBS1. In another particularly suitableembodiment of the substrate protein, the endogenous protease cleavagesequence can be located, for example, between about amino acid position142 to about amino acid position 165 when the substrate protein is RIN4.

Methods for synthesizing nucleic acid molecules are well known in theart, such as cloning and digestion of the appropriate sequences, as wellas direct chemical synthesis (e.g., ink-jet deposition andelectrochemical synthesis). Methods of cloning nucleic acid moleculesare described, for example, in Ausubel et al. (1995), supra; Copeland etal. (2001) Nat. Rev. Genet. 2:769-779; PCR Cloning Protocols, 2nd ed.(Chen & Janes eds., Humana Press 2002); and Sambrook & Russell (2001),supra. Methods of direct chemical synthesis of nucleic acid moleculesinclude, but are not limited to, the phosphotriester methods of Reese(1978) Tetrahedron 34:3143-3179 and Narang et al. (1979) MethodsEnzymol. 68:90-98; the phosphodiester method of Brown et al. (1979)Methods Enzymol. 68:109-151; the diethylphosphoramidate method ofBeaucage et al. (1981) Tetrahedron Lett. 22:1859-1862; and the solidsupport methods of Fodor et al. (1991) Science 251:767-773; Pease et al.(1994) Proc. Natl. Acad. Sci. USA 91:5022-5026; and Singh-Gasson et al.(1999) Nature Biotechnol. 17:974-978; as well as U.S. Pat. No.4,485,066. See also, Peattie (1979) Proc. Natl. Acad. Sci. USA76:1760-1764; as well as EP Patent No. 1 721 908; Int'l PatentApplication Publication Nos. WO 2004/022770 and WO 2005/082923; USPatent Application Publication No. 2009/0062521; and U.S. Pat. Nos.6,521,427; 6,818,395 and 7,521,178.

In addition to the full-length nucleotide sequence of a nucleic acidmolecule encoding a modified substrate protein, it is intended that thenucleic acid molecule can be a fragment or variant thereof that iscapable of functioning as a substrate. For nucleotide sequences,“fragment” means a portion of a nucleotide sequence of a nucleic acidmolecule, for example, a portion of the nucleotide sequence encoding amodified substrate protein. Fragments of a nucleotide sequence mayretain the biological activity of the reference nucleic acid molecule.For example, less than the entire sequence disclosed in SEQ ID NO:5 or 7can be used and will encode a modified substrate protein that interactswith a pathogen-specific protease and that retains its ability tointeract with its corresponding NB-LRR protein. Likewise, a fragment ofa nucleotide sequence encoding the modified substrate protein can beused if that fragment encodes a modified substrate protein thatinteracts with a pathogen-specific protease and that retains its abilityto interact with its corresponding NB-LRR protein. Alternatively,fragments of a nucleotide sequence that can be used as hybridizationprobes generally do not need to retain biological activity. Thus,fragments of the nucleic acid molecules can be at least about 10, 15,20, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850 or 900 nucleotides, or up to thenumber of nucleotides present in a full-length nucleic acid molecule.

A fragment of the nucleic acid molecule therefore can include afunctionally/biologically active portion, or it can include a fragmentthat can be used as a hybridization probe or PCR primer. A biologicallyactive portion of the nucleic acid molecule can be prepared by isolatingpart of the sequence of the nucleic acid molecule, operably linking thatfragment to a promoter, expressing the nucleotide sequence encoding theprotein, and assessing the amount or activity of the protein. Methods ofassaying protein expression are well known in the art. See, e.g., Chanet al. (1994) J. Biol. Chem. 269:17635-17641; Freyssinet & Thomas (1998)Pure & Appl. Chem. 70:61-66; and Kirby et al. (2007) Adv. Clin. Chem.44:247-292; as well as US Patent Application Publication Nos.2009/0183286 and 2009/0217424; and U.S. Pat. Nos. 7,294,711 and7,408,055. Likewise, kits for assaying protein expression arecommercially available, for example, from Applied Biosystems, Inc.(Foster City, Calif.), Caliper Life Sciences (Hopkinton, Mass.), Promega(Madison, Wis.), and SABiosciences (Frederick, Md.). Protein expressionalso can be assayed using other methods well known in the art,including, but not limited to, Western blot analysis, enzyme-linkedimmunosorbent assay, and the like. See, e.g., Sambrook & Russel (2001),supra. Moreover, methods of assaying pathogen-specific proteasesubstrate protein activity are well known in the art. See, DeYoung etal. (2012), supra.

For nucleotide sequences, “variant” means a substantially similarnucleotide sequence to a nucleotide sequence of a recombinant nucleicacid molecule as described herein, for example, a substantially similarnucleotide sequence encoding a modified substrate protein. Fornucleotide sequences, a variant comprises a nucleotide sequence havingdeletions (i.e., truncations) at the 5′ and/or 3′ end, deletions and/oradditions of one or more nucleotides at one or more internal sitescompared to the nucleotide sequence of the recombinant nucleic acidmolecules as described herein; and/or substitution of one or morenucleotides at one or more sites compared to the nucleotide sequence ofthe recombinant nucleic acid molecules described herein. One of skill inthe art understands that variants are constructed in a manner tomaintain the open reading frame.

Conservative variants include those nucleotide sequences that, becauseof the degeneracy of the genetic code (see, Table 2), result in afunctionally active modified substrate protein as described herein.Naturally occurring allelic variants can be identified by usingwell-known molecular biology techniques such as, for example, polymerasechain reaction (PCR) and hybridization techniques. Variant nucleotidesequences also can include synthetically derived sequences, such asthose generated, for example, by site-directed mutagenesis but whichstill provide a functionally active modified substrate protein.Generally, variants of a nucleotide sequence of the recombinant nucleicacid molecules as described herein will have at least about 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the nucleotide sequence of the recombinant nucleicacid molecules as determined by sequence alignment programs andparameters as described elsewhere herein.

When making recombinant nucleic acid molecules as described herein andvariants thereof, one of skill in the art can be further guided byknowledge of redundancy in the genetic code as shown below in Table 2.

TABLE 2 Redundancy in Genetic Code. Residue Triplet Codons Encoding theResidue Ala (A) GCU, GCC, GCA, GCG Arg (R) CGU, CGC, CGA, CGG, AGA, AGGAsn (N) AAU, AAC Asp (D) GAU, GAC Cys (C) UGU, UGC Gln (Q) CAA, CAG Glu(E) GAA, GAG Gly (G) GGU, GGC, GGA, GGG His (H) CAU, CAC Ile (I) AUU,AUC, AUA Leu (L) UUA, UUG, CUU, CUC, CUA, CUG Lys (K) AAA, AAG Met (M)AUG Phe (F) UUU, UUC Pro (P) CCU, CCC, CCA, CCG Ser (S) UCU, UCC, UCA,UCG, AGU, AGC Thr (T) ACU, ACC, ACA, ACG Trp (W) UGG Tyr (Y) UAU, UACVal (V) GUU, GUC, GUA, GUG START AUG STOP UAG, UGA, UAA

Deletions, insertions and/or substitutions of the nucleotide sequence ofthe recombinant nucleic acid molecules are not expected to produceradical changes in their characteristics. However, when it is difficultto predict the exact effect of the substitution, deletion or insertionin advance of doing so, one of skill in the art will appreciate that theeffect can be evaluated by expression assays.

Variant nucleic acid molecules also encompass nucleotide sequencesderived from a mutagenic and recombinogenic procedure such as DNAshuffling. With such a procedure, the nucleotide sequences of therecombinant nucleic acid molecules described herein can be manipulatedto create a new nucleic acid molecule possessing the desired properties.In this manner, libraries of recombinant nucleic acid molecules can begenerated from a population of related nucleic acid molecules comprisingsequence regions that have substantial sequence identity and can behomologously recombined in vitro or in vivo. For example, using thisapproach, sequence motifs encoding a domain of interest can be shuffledbetween the nucleic acid molecules described herein and other knownpromoters to obtain a new nucleic acid molecule with an improvedproperty such as increased promoter activity.

Methods of mutating and altering nucleotide sequences, as well as DNAshuffling, are well known in the art. See, Crameri et al. (1997) NatureBiotech. 15:436-438; Crameri et al. (1998) Nature 391:288-291; Kunkel(1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987)Methods in Enzymol. 154:367-382; Moore et al. (1997) J. Mol. Biol.272:336-347; Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Zhang et al. (1997) Proc. Natl. Acad.Sci. USA 94:4504-4509; and Techniques in Molecular Biology (Walker &Gaastra eds., MacMillan Publishing Co. 1983) and the references citedtherein; as well as U.S. Pat. Nos. 4,873,192; 5,605,793 and 5,837,458.As such, the nucleic acid molecules as described herein can have manymodifications.

Variants of the recombinant nucleic acid molecules described herein alsocan be evaluated by comparing the percent sequence identity between thepolypeptide encoded by a variant and the polypeptide encoded by areference nucleic acid molecule. Thus, for example, an isolated nucleicacid molecule can be one that encodes a polypeptide with a given percentsequence identity to the polypeptide of interest. Percent sequenceidentity between any two polypeptides can be calculated using sequencealignment programs and parameters described elsewhere herein. Where anygiven pair of polynucleotides of the present disclosure is evaluated bycomparison of the percent sequence identity shared by the twopolypeptides they encode, the percent sequence identity between the twoencoded polypeptides can be at least about 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

Determining percent sequence identity between any two sequences can beaccomplished using a mathematical algorithm. Non-limiting examples ofsuch mathematical algorithms include, but are not limited to, thealgorithm of Myers & Miller (1988) CABIOS 4:11-17; the local alignmentalgorithm of Smith et al. (1981) Adv. Appl. Math. 2:482-489; the globalalignment algorithm of Needleman & Wunsch (1970) J. Mol. Biol.48:443-453; the search-for-local alignment method of Pearson & Lipman(1988) Proc. Natl. Acad. Sci. USA 85:2444-2448; the algorithm of Karlin& Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified asin Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

The present disclosure therefore includes recombinant nucleic acidmolecules having a nucleotide sequence that encodes a modified substrateprotein of a pathogen-specific protease, where the modified substrateprotein has a heterologous protease recognition sequence and can beincorporated into nucleic acid constructs such as expression cassettesand vectors.

Nucleic Acid Constructs

Compositions of the present disclosure also include nucleic acidconstructs, such as expression cassettes or vectors, having plantpromoters operably linked with a nucleic acid molecule that encodes asubstrate protein of a pathogen-specific protease with a heterologouspathogen-specific protease recognition sequence for use in transformingplant cells, plant parts and plants. In addition, the constructs caninclude a nucleic acid molecule that encodes a NB-LRR protein,particularly when such NB-LRR protein is not native/not endogenous tothe plant cell, plant part or plant to be transformed.

As used herein, “nucleic acid construct” means an oligonucleotide orpolynucleotide composed of deoxyribonucleotides, ribonucleotides orcombinations thereof having incorporated therein the nucleotidesequences described herein. The nucleotide construct can be used fortransforming organisms such as plants. In this manner, plant promotersoperably linked to a nucleotide sequence for a modified substrateprotein of a pathogen-specific protease as described herein are providedin nucleic acid constructs for expression in a plant cell, plant part orplant.

As used herein, “expression cassette” means a nucleic acid moleculehaving at least a control sequence operably linked to a coding sequence.

As used herein, “operably linked” means that the elements of theexpression cassette are configured so as to perform their usualfunction. Thus, control sequences (i.e., promoters) operably linked to acoding sequence are capable of effecting expression of the codingsequence. The control sequences need not be contiguous with the codingsequence, so long as they function to direct the expression thereof.Thus, for example, intervening untranslated, yet transcribed, sequencescan be present between a promoter and a coding sequence, and thepromoter sequence still can be considered “operably linked” to thecoding sequence.

As used herein, a “coding sequence” or “coding sequences” means asequence that encodes a particular polypeptide, and is a nucleotidesequence that is transcribed (in the case of DNA) and translated (in thecase of mRNA) into a polypeptide in vitro or in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at a 5′ (amino) terminusand a translation stop codon at a 3′ (carboxy) terminus. A codingsequence can include, but is not limited to, viral nucleic acidsequences, cDNA from prokaryotic or eukaryotic mRNA, genomic DNAsequences from prokaryotic or eukaryotic DNA, and even synthetic DNAsequences. A transcription termination sequence will usually be located3′ to the coding sequence. Examples of coding sequences for use hereininclude nucleotide sequence that encodes a modified substrate protein ofa pathogen-specific protease, a NB-LRR protein or both.

As used herein, “control sequence” or “control sequences” meanspromoters, polyadenylation signals, transcription and translationtermination sequences, upstream regulatory domains, origins ofreplication, internal ribosome entry sites (“IRES”), enhancers, and thelike, which collectively provide for replication, transcription andtranslation of a coding sequence in a recipient host cell. Not all ofthese control sequences need always be present so long as the selectedcoding sequence is capable of being replicated, transcribed andtranslated in an appropriate host cell.

As used herein, a “promoter” means a nucleotide region comprising anucleic acid (i.e., DNA) regulatory sequence, wherein the regulatorysequence is derived from a gene or synthetically created that is capableof binding RNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence. A number of promoters can be used in theexpression cassette, including the native promoter of the modifiedsubstrate protein or NB-LRR protein.

Alternatively, promoters can be selected based upon a desired outcome.Such promoters include, but are not limited to, “constitutive promoters”(where expression of a polynucleotide sequence operably linked to thepromoter is unregulated and therefore continuous), “inducible promoters”(where expression of a polynucleotide sequence operably linked to thepromoter is induced by an analyte, cofactor, regulatory protein, etc.),and “repressible promoters” (where expression of a polynucleotidesequence operably linked to the promoter is repressed by an analyte,cofactor, regulatory protein, etc.).

As used herein, “plant promoter” means a promoter that drives expressionin a plant such as a constitutive, inducible (e.g., chemical-,environmental-, pathogen- or wound-inducible), repressible,tissue-preferred or other promoter for use in plants.

Examples of constitutive promoters include, but are not limited to, therice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406;and U.S. Pat. No. 5,641,876), the CaMV 19S promoter (Lawton et al.(1987) Plant Mol. Biol. 9:315-324), the CaMV 35S promoter (Odell et al.(1985) Nature 313:810-812), the nos promoter (Ebert et al. (1987) Proc.Natl. Acad. Sci. USA 84:5754-5749), the Adh promoter (Walker et al.(1987) Proc. Natl. Acad. Sci. USA 84:6624-6628), the sucrose synthasepromoter (Yang & Russell (1990) Proc. Natl. Acad. Sci. USA87:4144-4148), the ubiquitin promoters, and the like. See also, U.S.Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142 and 6,177,611.

Examples of chemical-inducible promoters include, but are not limitedto, the maize Tn2-2 promoter, which is activated by benzenesulfonamideherbicide safeners; the maize GST promoter, which is activated byhydrophobic electrophilic compounds that are used as pre-emergentherbicides; and the tobacco PR-1a promoter, which is activated bysalicylic acid. Other chemical-inducible promoters of interest includesteroid-responsive promoters (e.g., the glucocorticoid-induciblepromoters in Aoyama & Chua (1997) Plant J. 11:605-612; McNellis et al.(1998) Plant J. 14:247-257; and Schena et al. (1991) Proc. Natl. Acad.Sci. USA 88:10421-10425); tetracycline-inducible andtetracycline-repressible promoters (Gatz et al. (1991) Mol. Gen. Genet.227:229-237; as well as U.S. Pat. Nos. 5,814,618 and 5,789,156); ABA-and turgor-inducible promoters, the auxin-binding protein gene promoter(Schwob et al. (1993) Plant J. 4:423-432), the UDP glucose flavonoidglycosyl-transferase gene promoter (Ralston et al. (1988) Genetics119:185-187), the MPI proteinase inhibitor promoter (Cordero et al.(1994) Plant J. 6:141-150), and the glyceraldehyde-3-phosphatedehydrogenase gene promoter (Kohler et al. (1995) Plant Mol. Biol.29:1293-1298; Martinez et al. (1989) J. Mol. Biol. 208:551-565; andQuigley et al. (1989) J. Mol. Evol. 29:412-421). Also included are thebenzene sulphonamide-inducible (U.S. Pat. No. 5,364,780) andalcohol-inducible (Int'l Patent Application Publication Nos. WO 97/06269and WO 97/06268) systems and glutathione S-transferase promoters.Chemical-inducible promoters therefore can be used to modulate theexpression of a nucleotide sequence of interest in a plant by applyingan exogenous chemical regulator. Depending upon the objective, thepromoter can be a chemical-inducible promoter, whereby application ofthe chemical induces gene expression, or a chemical-repressiblepromoter, whereby application of the chemical represses gene expression.See also, Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89.

Other inducible promoters include promoters from genes induciblyregulated in response to environmental stress or stimuli such asdrought, pathogens, salinity and wounds. See, Graham et al. (1985) J.Biol. Chem. 260:6555-6560; Graham et al. (1985) J. Biol. Chem.260:6561-6564; and Smith et al. (1986) Planta 168:94-100.Wound-inducible promoters include the metallocarboxypeptidase-inhibitorprotein promoter (Graham et al. (1981) Biochem. Biophys. Res. Comm.101:1164-1170).

Examples of tissue-preferred promoters include, but are not limited to,the rbcS promoter, the ocs, nos and mas promoters that have higheractivity in roots or wounded leaf tissue, a truncated (−90 to +8) 35Spromoter that directs enhanced expression in roots, an α-tubulin genepromoter that directs expression in roots, as well as promoters derivedfrom zein storage protein genes that direct expression in endosperm.Additional examples of tissue-preferred promoters include, but are notlimited to, the promoters of genes encoding the seed storage proteins(e.g., β-conglycinin, cruciferin, napin and phaseolin), zein or oil bodyproteins (e.g., oleosin), or promoters of genes involved in fatty acidbiosynthesis (e.g., acyl carrier protein, stearoyl-ACP desaturase andfatty acid desaturases (e.g., fad 2-1)), and promoters of other genesexpressed during embryo development (e.g., Bce4; Kridl et al. (1991)Seed Sci. Res. 1:209-219). Further examples of tissue-specific promotersinclude, but are not limited to, the lectin promoter (Lindstrom et al.(1990) Dev. Genet. 11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res.138:87-98), the corn alcohol dehydrogenase 1 promoter (Dennis et al.(1984) Nucleic Acids Res. 12:3983-4000; and Vogel et al. (1989) J. Cell.Biochem. 13: Part D, M350 (Abstract)), corn light harvesting complex(Bansal et al. (1992) Proc. Natl. Acad. Sci. USA 89:3654-3658; andSimpson (1986) Science 233:34-380), corn heat shock protein (Odell etal. (1985) Nature 313:810-812; and Rochester et al. (1986) EMBO J.5:451-458), the pea small subunit RuBP carboxylase promoter (Cashmore,“Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphatecarboxylase” 29-38 In: Gen. Eng. of Plants (Plenum Press 1983); andPoulsen et al. (1986) Mol. Gen. Genet. 205:193-200), the Ti plasmidmannopine synthase promoter (Langridge et al. (1989) Proc. Natl. Acad.Sci. USA 86:3219-3223), the Ti plasmid nopaline synthase promoter(Langridge et al. (1989), supra), the petunia chalcone isomerasepromoter (van Tunen et al. (1988) EMBO J. 7:1257-1263), the bean glycinerich protein 1 promoter (Keller et al. (1989) Genes Dev. 3:1639-1646),the truncated CaMV 35s promoter (Odell et al. (1985), supra), the potatopatatin promoter (Wenzler et al. (1989) Plant Mol. Biol. 13:347-354),the root cell promoter (Yamamoto et al. (1990) Nucleic Acids Res.18:7449), the maize zein promoter (Langridge et al. (1983) Cell34:1015-1022; Kriz et al. (1987) Mol. Gen. Genet. 207:90-98; Reina etal. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic AcidsRes. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354), theglobulin-1 gene (Belanger et al. (1991) Genetics 129:863-872), theα-tubulin, cab promoter (Sullivan et al. (1989) Mol. Gen. Genet.215:431-440), the PEPCase promoter (Hudspeth & Grula (1989) Plant Mol.Biol. 12:579-589), the R gene complex-associated promoters (Chandler etal. (1989) Plant Cell 1:1175-1183), and the chalcone synthase promoters(Franken et al. (1991) EMBO J. 10:2605-2612). See also, Canevascini etal. (1996) Plant Physiol. 112:513-524; Guevara-Garcia et al. (1993)Plant J. 4:495-505; Hansen et al. (1997) Mol. Gen. Genet. 254:337-343;Kawamata et al. (1997) Plant Cell Physiol. 38:792-803; Lam (1994)Results Probl. Cell Differ. 20:181-196; Matsuoka et al. (1993) Proc.Natl. Acad. Sci. USA 90:9586-9590; Orozco et al. (1993) Plant Mol. Biol.23:1129-1138; Rinehart et al. (1996) Plant Physiol. 112:1331-1341;Russell et al. (1997) Transgenic Res. 6:157-168; Van Camp et al. (1996)Plant Physiol. 112:525-535; Yamamoto et al. (1994) Plant Cell Physiol.35:773-778; and Yamamoto et al. (1997) Plant J. 12:255-265.

In some instances, the tissue-preferred promoter can be a leaf-preferredpromoter. See, Gan et al. (1995) Science 270:1986-1988; Gotor et al.(1993) Plant J. 3:509-518; Kwon et al. (1994) Plant Physiol.105:357-367; Matsuoka et al. (1993), supra; Orozco et al. (1993), supra;Yamamoto et al. (1994), supra; and Yamamoto et al. (1997), supra.

In some instances, the tissue-preferred promoter can be a root-preferredpromoter. See, Capana et al. (1994) Plant Mol. Biol. 25:681-691 (rolBpromoter); Hire et al. (1992) Plant Mol. Biol. 20:207-218 (soybeanroot-specific glutamine synthetase gene); Keller & Baumgartner (1991)Plant Cell 3:1051-1061 (root-specific control element in the GRP 1.8gene of French bean); Kuster et al. (1995) Plant Mol. Biol. 29:759-772(VfENOD-GRP3 gene promoter) Miao et al. (1991) Plant Cell 3:11-22(full-length cDNA clone encoding cytosolic glutamine synthetase (GS),which is expressed in roots and root nodules of soybean); and Sanger etal. (1990) Plant Mol. Biol. 14:433-443 (root-specific promoter of themannopine synthase (MAS) gene of A. tumefaciens); see also, U.S. Pat.Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732;and 5,023,179. Likewise, Bogusz et al. (1990) Plant Cell 2:633-641describes two root-specific promoters isolated from hemoglobin genesfrom the nitrogen-fixing nonlegume Parasponia andersonii and the relatednon-nitrogen-fixing nonlegume Trema tomentosa. Leach & Aoyagi (1991)Plant Sci. 79:69-76 describes an analysis of the promoters of the highlyexpressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes.Teeri et al. (1989) EMBO J. 8:343-335 describes a gene fusion to lacZ toshow that the Agrobacterium T-DNA gene encoding octopine synthase isespecially active in the epidermis of the root tip and that the TR2′gene is root specific in the intact plant and stimulated by wounding inleaf tissue.

In some instances, the tissue-preferred promoter can be a seed-preferredpromoter, which includes both “seed-specific” promoters (i.e., promotersactive during seed development such as promoters of seed storageproteins) and “seed-germinating” promoters (i.e., promoters activeduring seed germination). See, Thompson et al. (1989) BioEssays10:108-113. Examples of seed-preferred promoters include, but are notlimited to, the Cim1 promoter (cytokinin-induced message); the cZ19B1promoter (maize 19 kDa zein); the myo-inositol-1-phosphate synthase(milps) promoter (Int'l Patent Application Publication No. WO 00/11177;and U.S. Pat. No. 6,225,529); the γ-zein promoter; and the globulin 1(Glb-1) promoter. For monocots, seed-specific promoters include, but arenot limited to, promoters from maize 15 kDa zein, 22 kDa zein, 27 kDazein, γ-zein, waxy, shrunken 1, shrunken 2 and Glb-1. See also, Int'lPatent Application Publication No. WO 00/12733, which disclosesseed-preferred promoters from end1 and end2 genes. For dicots,seed-specific promoters include, but are not limited to, promoters frombean 3-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin andpea vicilin (Czako et al. (1992) Mol. Gen. Genet. 235:33-40). See also,U.S. Pat. No. 5,625,136.

In some instances, the tissue-preferred promoter can be astalk-preferred promoter. Examples of stalk-preferred promoters include,but are not limited to, the maize MS8-15 gene promoter (Int'l PatentApplication Publication No. WO 98/00533; and U.S. Pat. No. 5,986,174),and the promoters disclosed in Graham et al. (1997) Plant Mol. Biol.33:729-735.

In some instances, the tissue-preferred promoter can be a vasculartissue-preferred promoter. For example, a vascular tissue-preferredpromoter can be used to express the modified substrate protein inpolypexylem and phloem tissue. Examples of vascular tissue-preferredpromoters include, but are not limited to, the Prunus serotina prunasinhydrolase gene promoter (Int'l Patent Application Publication No. WO03/006651), and the promoters disclosed in U.S. Pat. No. 6,921,815.

As an alternative to the promoters listed above, in some instances a lowlevel of expression is desired and can be achieved by using a weakpromoter. As used herein, “weak promoter” means a promoter that drivesexpression of a coding sequence at a low level. As used herein, “lowlevel” means at levels of about 1/1000 transcripts to about 1/100,000transcripts to about 1/500,000 transcripts. Alternatively, it isrecognized that weak promoter also encompasses promoters that areexpressed in only a few cells and not in others to give a total lowlevel of expression. Where a promoter is expressed at unacceptably highlevels, portions of the promoter sequence can be deleted or modified todecrease expression levels.

Examples of weak constitutive promoters include, but are not limited to,the core promoter of the Rsyn7 promoter (Int'l Patent ApplicationPublication No. WO 99/43838 and U.S. Pat. No. 6,072,050), the core 35SCaMV promoter, and the like. Other weak constitutive promoters aredescribed, for example, in U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and6,177,611.

Weak promoters can be used when designing expression cassettes forNB-LRR proteins, as NB-LRR genes preferably are constitutively expressedat low levels because high levels can lead to cell death in the absenceof pathogens.

The expression cassette can include other control sequences 5′ to thecoding sequence. For example, the expression cassette can include a 5′leader sequence, which can act to enhance translation. Examples of 5′leader sequences include, but are not limited to, picornavirus leaders(e.g., encephalomyocarditis virus (EMCV) leader; Elroy-Stein et al.(1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders(e.g., tobacco etch virus (TEV) leader; Gallie et al. (1995) Gene165:233-238); maize dwarf mosaic virus (MDMV) leader (Allison et al.(1986) Virology 154:9-20); human immunoglobulin heavy-chain bindingprotein (BiP; Macejak et al. (1991) Nature 353:90-94); untranslatedleader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 94;Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus (TMV)leader (Gallie et al., “Eukaryotic viral 5′-leader sequences act astranslational enhancers in eukaryotes and prokaryotes” 237-256 In:Molecular Biology of RNA (Cech ed., Liss 1989)); and maize chloroticmottle virus (MCMV) leader (Lommel et al. (1991) Virology 81:382-385).See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; andGallie (1996) Plant Mol. Biol. 32:145-158. Other methods or sequencesknown to enhance translation also can be used, for example, introns, andthe like.

The expression cassette also can include a coding sequence for themodified substrate protein of the pathogen-specific protease and/orNB-LRR protein. As discussed above, the modified substrate proteinincludes a heterologous protease recognition sequence. The heterologousprotease recognition sequence can be located within, for example, anexposed loop of the substrate protein. As noted above, nucleic and aminoacid sequences are well known in the art for many protease recognitionsequences that can be inserted into the substrate protein such as PBS1.In addition, nucleic and amino acid sequences are known in the art forvarious NB-LRR proteins. These sequences can be used when constructingthe expression cassette(s).

For example, the coding sequence can be SEQ ID NO:5 (modified PBS1having an AvrRpt2 protease recognition sequence) operably linked to thenative PBS1 promoter (SEQ ID NO:9). Alternatively, the coding sequencecan be SEQ ID NO:7 (modified PBS1 having a TEV protease recognitionsequence) operably linked to the native PBS1 promoter. Likewise, thecoding sequence can include a NB-LRR protein such as RPS5 when themodified substrate protein is based upon PBS1 (or RPS2 when the modifiedsubstrate protein is based upon RIN4).

The control sequence(s) and/or the coding sequence therefore can benative/analogous to the host cell or to each other. Alternatively, thecontrol sequence(s) and/or coding sequence can be heterologous to thehost cell or to each other. As used herein, “heterologous” means asequence that originates from a foreign species, or, if from the samespecies, is substantially modified from its native form in compositionand/or genomic locus by deliberate human intervention. For example, apromoter operably linked to a heterologous polynucleotide is from aspecies different from the species from which the polynucleotide wasderived, or, if from the same/analogous species, one or both aresubstantially modified from their original form and/or genomic locus, orthe promoter is not the native promoter for the operably linkedpolynucleotide.

The expression cassette also can include a transcriptional and/ortranslational termination region that is functional in plants. Thetermination region can be native with the transcriptional initiationregion (i.e., promoter), can be native with the operably linked codingsequence, can be native with the plant of interest, or can be derivedfrom another source (i.e., foreign or heterologous to the promoter, thecoding sequence, the plant host cell, or any combination thereof).Termination regions are typically located downstream (3′-direction) fromthe coding sequence. Termination regions include, but are not limitedto, the potato proteinase inhibitor (Pin II) gene or the Ti-plasmid ofA. tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See e.g., Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144;Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639; Mogen et al. (1990)Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Proudfoot(1991) Cell 64:671-674; and Sanfacon et al. (1991) Genes Dev. 5:141-149.

The expression cassette also can include one or more linkers. As usedherein, “linker” means a nucleotide sequence that functions to link oneelement of the expression cassette with another without otherwisecontributing to the transcription or translation of a nucleotidesequence of interest when present in the expression cassette. The linkercan include plasmid sequences, restriction sequences and/or sequences ofa 5′-untranslated region (5′-UTR). Alternatively, the linker further caninclude nucleotide sequences encoding the additional amino acid residuesthat naturally flank the heterologous protease recognition sequence inthe substrate protein from which it was isolated. The length andsequence of the linker can vary and can be about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1000 nucleotides or greater in length.

Just as expression of the modified substrate protein and/or NB-LRRprotein can be targeted to specific tissues or cell types by appropriateuse of promoters, it also can be targeted to different locations withina cell of a plant host by appropriate use of signal and/or targetingpeptide sequences. Unlike a promoter, which acts at the transcriptionallevel, signal and/or targeting peptide sequences are part of the initialtranslation product. Therefore, the expression cassette also can includea signal and/or targeting peptide sequence. Examples of such sequencesinclude, but are not limited to, the transit peptide for the acylcarrier protein, the small subunit of RUBISCO, plant EPSP synthase, andthe like. See, Archer et al. (1990) J. Bioenerg. Biomemb. 22:789-810;Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Daniell (1999) Nat.Biotech. 17:855-856; de Castro Silva Filho et al. (1996) Plant Mol.Biol. 30:769-780; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968;Lamppa et al. (1988) J. Biol. Chem. 263:14996-14999; Lawrence et al.(1997) J. Biol. Chem. 272:20357-20363; Romer et al. (1993) Biochem.Biophys. Res. Commun. 196:1414-1421; Schmidt et al. (1993) J. Biol.Chem. 268:27447-27457; Schnell et al. (1991) J. Biol. Chem.266:3335-3342; Shah et al. (1986) Science 233:478-481; Von Heijne et al.(1991) Plant Mol. Biol. Rep. 9:104-126; and Zhao et al. (1995) J. Biol.Chem. 270:6081-6087; as well as U.S. Pat. No. 6,338,168.

It may be desirable to locate the modified substrate protein and/orNB-LRR protein on specific plant membranes such as the plasma membraneor tonoplast membrane. This can be accomplished, for example, by addingspecific amino acid sequences to the N-terminus of these proteins byadding specific sequences to the expression cassette as described inRaikhel & Chrispeels, “Protein sorting and vesicle traffic” In:Biochemistry and Molecular Biology of Plants (Buchanan et al. eds.,American Society of Plant Physiologists 2000). See also, Denecke et al.(1992) EMBO J. 11:2345-2355; Denecke et al. (1993) J. Exp. Bot.44:213-221; Gomord et al. (1996) Plant Physiol. Biochem. 34:165-181;Lehmann et al. (2001) Plant Physiol. 127:436-449; Munro & Pelham (1986)Cell 46:291-300; Munro & Pelham (1987) Cell 48:899-907; Vitale et al.(1993) J. Exp. Bot. 44:1417-1444; and Wandelt et al. (1992) Plant J.2:181-192.

Additional guidance on subcellular targeting of proteins in plants canbe found, for example, in Bruce (2001) Biochim Biophys Acta 1541:2-21;Emanuelsson et al. (2000) J. Mol. Biol. 300:1005-1016; Emanuelsson & vonHeijne (2001) Biochim Biophys Acta 1541:114-119; Hadlington & Denecke(2000) Curr. Opin. Plant Biol. 3:461-468; Nicchitta (2002) Curr. Opin.Cell Biol. 14:412-416; and Silva-Filho (2003) Curr. Opin. Plant Biol.6:589-595.

The expression cassette also can include nucleotide sequences encodingagronomic and pesticidal polypeptides, and the like. Such sequences canbe stacked with any combination of nucleotide sequences to create plantcells, plants parts and plants with a desired phenotype. For example,the nucleic acid molecule encoding modified substrate protein and/orNB-LRR protein can be stacked with nucleotide sequences encoding apesticidal polypeptide such as a δ-endotoxin. The combinations generatedalso can include multiple copies of any one of the nucleotide sequencesof interest. Examples of other nucleotide sequences of interest include,but are not limited to, sequences encoding for high oil (U.S. Pat. No.6,232,529); balanced amino acids (hordothionins; U.S. Pat. Nos.5,703,409; 5,885,801; 5,885,802 and 5,990,389); barley high lysine(Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and Int'l PatentApplication Publication No. WO 98/20122); high methionine proteins(Pedersen et al. (1986) J. Biol. Chem. 261:6279-6284; Kirihara et al.(1988) Gene 71:359-370; and Musumura et al. (1989) Plant Mol. Biol.12:123-130); increased digestibility (modified storage proteins; U.S.Pat. No. 6,858,778); and thioredoxins (U.S. Pat. No. 7,009,087).

The nucleotide sequence encoding the modified substrate protein and/orNB-LRR disease resistance protein also can be stacked with nucleotidesequences encoding polypeptides for herbicide resistance (e.g.,glyphosate or HPPD resistance; see, e.g., EPSPS genes, GAT genes (Int'lPatent Application Publication Nos. WO 02/36782 and WO 03/092360; and USPatent Application Publication No. 2004/0082770); lectins (Van Damme etal. (1994) Plant Mol. Biol. 24:825-830); fumonisin detoxification (U.S.Pat. No. 5,792,931); acetolactate synthase (ALS) mutants that lead toherbicide resistance such as the S4 and/or Hra mutations; inhibitors ofglutamine synthase such as phosphinothricin or basta (e.g., bar gene);modified starches (ADPG pyrophosphorylases (AGPase), starch synthases(SS), starch branching enzymes (SBE) and starch debranching enzymes(SDBE)); and polymers or bioplastics (U.S. Pat. No. 5,602,321);beta-ketothiolase, polyhydroxybutyrate synthase and acetoacetyl-CoAreductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847).

The nucleotide sequence encoding the modified substrate protein and/orNB-LRR disease resistance protein also can be stacked with nucleotidesequences encoding for agronomic traits such as male sterility (U.S.Pat. No. 5,583,210), stalk strength, flowering time or transformationtechnology traits such as cell cycle regulation or gene targeting (Int'lPatent Application Publication Nos. and WO 99/25821; WO 99/61619 and WO00/17364).

These stacked combinations can be created by any method including, butnot limited, to cross breeding plants by any conventional or TopCross™methodology (DuPont Specialty Grains; Des Moines, Iowa), zinc fingernucleases (ZFNs), transcription activator-like effector nucleases(TALENs) or other genetic transformation. If the traits are stacked bygenetically transforming the plants, the nucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separate expressioncassettes (trans) or contained on the same transformation cassette(cis). Expression of the sequences can be driven by the same promoter orby different promoters. In certain instances, it may be desirable tointroduce an expression cassette that will suppress the expression ofthe polynucleotide of interest. This may be combined with anycombination of other suppression cassettes or overexpression cassettesto generate the desired combination of traits in the plant. It isfurther recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, Int'l Patent Application Publication Nos. WO 99/25821; WO 99/25840;WO 99/25853; WO 99/25854 and WO 99/25855.

In addition to the above, it is contemplated that the nucleic acidconstructs can be used in the form of a system, particularly when usedin plant cells, plant parts and plants that lack a substrate protein ofa pathogen-specific protease and NB-LRR protein pair. Such systems caninclude one or more nucleic acid constructs, such as expressioncassettes or vectors, having a promoter that drives expression in aplant, plant part or plant cell operably linked to a coding sequence fora modified substrate protein of a pathogen-specific protease, where thesubstrate protein has a heterologous protease recognition sequence, anda sequence for a promoter that drives expression in a plant, plant partor plant cell operably linked to a coding sequence for a NB-LRR protein.The promoters can be the same or can be distinct. For example, the firstpromoter can be an inducible promoter and the second promoter can be aconstitutive promoter, especially a weak constitutive promoter.Alternatively, both the first and second promoters can be inducible,repressible or constitutive. The NB-LRR protein can associate with, andcan be activated by, the modified substrate. Such systems therefore canbe used to provide the protein pair to a plant cell, plant part or plantthat does not natively express the protein pair.

Alternatively, the system can include a first nucleic acid constructhaving nucleotide sequence for a promoter that drives expression in aplant cell, plant part or plant operably linked to a coding sequence fora modified substrate protein of a pathogen-specific protease asdescribed herein, and a second nucleic acid construct having anucleotide sequence for a promoter that drives expression in a plantcell, plant part or plant operably linked to a coding sequence for aNB-LRR protein.

Additional nucleic acid constructs also can be included in the system,where each construct has a nucleotide sequence that encodes a distinctmodified substrate protein, each having a heterologous recognitionsequence for a separate pathogen-specific protease. Although eachmodified substrate protein has a heterologous recognition sequencedistinct from one another, each can associate with, and can activate,the NB-LRR protein. For example, the nucleic acid construct(s) canencode (1) a PBS1 in which its endogenous AvrPphB cleavage site (SEQ IDNO:1) is replaced with a heterologous AvrRpt2 cleavage site (SEQ IDNO:2), (2) a PBS1 in which its endogenous AvrPphB cleavage site (SEQ IDNO:1) is replaced with a heterologous TEV protease cleavage site (SEQ IDNO:4) and/or (3) a PBS1 in which its endogenous AvrPphB cleavage site(SEQ ID NO:1) is replaced with a heterologous HopN1 cleavage site (SEQID NO:3). Similarly, the nucleic acid construct(s) can encode (1) a PBS1in which its endogenous AvrPphB cleavage site (SEQ ID NO:1) is replacedwith a heterologous SMV cleavage site (SEQ ID NO:27) and/or (2) a PBS1in which its endogenous AvrPphB cleavage site (SEQ ID NO:1) is replacedwith a heterologous BPMV protease cleavage site (SEQ ID NO:28). Althougheach of these modified substrate proteins would be targets for distinctpathogen-specific proteases, all would be expected to associate with andactivate a RPS5 protein. In another example, the nucleic acidconstruct(s) can encode (1) a RIN4 in which its endogenous AvrRpt2cleavage site (SEQ ID NO:2) is replaced with a heterologous AvrPphBcleavage site (SEQ ID NO:1). (2) a RIN4 in which its endogenous AvrRpt2cleavage site (SEQ ID NO:2) is replaced with a heterologous TEV proteasecleavage site (SEQ ID NO:4) and/or (3) a RIN4 in which its endogenousAvrRpt2 cleavage site (SEQ ID NO:2) is replaced with a heterologousHopN1 cleavage site (SEQ ID NO:3). Similarly, the nucleic acidconstruct(s) can encode (1) a RIN4 in which its endogenous AvrRpt2cleavage site (SEQ ID NO:2) is replaced with a heterologous SMV cleavagesite (SEQ ID NO:27) and/or (2) a RIN4 in which its endogenous AvrRpt2cleavage site (SEQ ID NO:2) is replaced with a heterologous BPMVprotease cleavage site (SEQ ID NO:28). Although each of these modifiedsubstrate proteins would be targets for distinct pathogen-specificproteases, all would be expected to associate with and activate a RPS2protein.

As such, the first nucleic acid construct can encode more than onemodified substrate protein, where each modified substrate protein has aheterologous recognition sequence distinct from one another and whereeach can associate with, and can activate, the NB-LRR protein.Alternatively, the second nucleic acid construct can encode one or moremodified substrate proteins, where each modified substrate protein has aheterologous recognition sequence distinct from one another and whereeach can associate with, and can activate, the NB-LRR protein. As above,the promoters can be the same or can be distinct. Such systems can beused to provide the protein pair to a plant cell, plant part or plantthat does not natively express the protein pair or can be used toprovide more than one modified substrate to a plant cell, plant part orplant.

Regardless of whether used as individual nucleic acid constructs orsystems, and where appropriate, the nucleotide sequences can beoptimized for increased expression in plants. That is, the nucleotidesequences can be synthesized using plant-preferred codons for improvedexpression. Methods for optimizing nucleotide sequences for expressionin plants are well known in the art. See, Campbell & Gowri (1990) PlantPhysiol. 92:1-11; Murray et al. (1989) Nucleic Acids Res. 17:477-498;and Wada et al. (1990) Nucl. Acids Res. 18:2367-2411; as well as U.S.Pat. Nos. 5,096,825; 5,380,831; 5,436,391; 5,625,136; 5,670,356 and5,874,304.

Likewise, additional sequence modifications are known to enhancenucleotide sequence expression in plants. These include elimination ofsequences encoding spurious polyadenylation signals, exon-intron splicesite signals, transposon-like repeats, and other such well-characterizedsequences that may be deleterious to gene expression. The G-C content ofthe sequence can be adjusted to levels average for a given cellularhost, as calculated by reference to known genes expressed in the hostplant. When possible, the nucleotide sequence can be modified to avoidpredicted hairpin secondary mRNA structures.

Methods of constructing expression cassettes are well known in the artand can be found, for example, in Balbás & Lorence, Recombinant GeneExpression: Reviews and Protocols, 2nd ed. (Humana Press 2004); Davis etal., Basic Methods in Molecular Biology (Elsevier Press 1986); Sambrook& Russell (2001), supra; Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Acid Probes (Elsevier1993); Ausubel et al. (1995), supra; as well as U.S. Pat. Nos.6,664,387; 7,060,491; 7,345,216 and 7,494,805.

The expression cassette therefore can include at least, in the directionof transcription (i.e., 5′ to 3′ direction), a plant promoter that isfunctional in a plant cell, plant part or plant operably linked to anucleotide sequence encoding a modified substrate protein having aheterologous protease recognition sequence. In some instances, theexpression cassette also can include a nucleotide sequence encoding aNB-LRR disease resistance protein.

To assist in introducing the nucleotide sequences of interest into theappropriate host cells, the expression cassette can be incorporated orligated into a vector. As used herein, “vector” means a replicon, suchas a plasmid, phage or cosmid, to which another nucleic acid segment maybe attached so as to bring about the replication of the attachedsegment. A vector is capable of transferring nucleic acid molecules tothe host cells. Bacterial vectors typically can be of plasmid or phageorigin.

Typically, the terms “vector construct,” “expression vector,” “geneexpression vector,” “gene delivery vector,” “gene transfer vector,” and“expression cassette” all refer to an assembly that is capable ofdirecting the expression of a sequence or gene of interest. Thus, theterms include cloning and expression vehicles.

Vectors typically contain one or a small number of restrictionendonuclease recognition sites where a nucleic acid molecule of interestcan be inserted in a determinable fashion without loss of essentialbiological function of the vector, as well as a selectable marker thatcan be used for identifying and selecting cells transformed with thevector.

A vector therefore can be capable of transferring nucleic acid moleculeto target cells (e.g., bacterial plasmid vectors, particulate carriersand liposomes). The selection of vector will depend upon the preferredtransformation technique and the target specie for transformation. Themost commonly used plant transformation vectors are binary vectorsbecause of their ability to replicate in intermediate host cells such asE. coli and A. tumefaciens. The intermediate host cells allow one toincrease the copy number of the cloning vector and/or to mediatetransformation of a different host cell. With an increased copy number,the vector containing the expression cassette of interest can beisolated in significant quantities for introduction into the desiredplant. General descriptions of plant vectors can be found, for example,in Gruber et al., “Vectors for plant transformation” 89-119 In: Methodsin Plant Molecular Biology & Biotechnology (Glich et al. eds., CRC Press1993). Examples of vectors for use with A. tumefaciens can be found, forexample, in U.S. Pat. No. 7,102,057.

Restriction enzymes can be used to introduce cuts into the targetnucleic acid molecule (e.g., nucleotide sequence encoding a modifiedsubstrate protein and/or NB-LRR protein) and the plasmid to facilitateinsertion of the target into the vector such as a plasmid. Moreover,restriction enzyme adapters such as EcoRl/Notl adapters can be added tothe target mRNA when the desired restriction enzyme sites are notpresent within it. Methods of adding restriction enzyme adapters arewell known in the art. See, Krebs et al. (2006) Anal. Biochem.350:313-315; and Lönneborg et al. (1995), supra. Likewise, kits foradding restriction enzyme sites are commercially available, for example,from Invitrogen (Carlsbad, Calif.).

Alternatively, viruses such as bacteriophages can be used as the vectorto deliver the target mRNA to competent host cells. Vectors can beconstructed using standard molecular biology techniques as described,for example, in Sambrook & Russell (2001), supra.

As noted above, selectable markers can be used to identify and selecttransformed plants, plant parts or plant host cells. Selectable markersinclude, but are not limited to, nucleotide sequences encodingantibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO), hygromycin phosphotransferase (HPT), aswell as nucleotide sequences encoding resistance to ampicillin,kanamycin, spectinomycin or tetracycline, and even nucleotide sequencesencoding herbicidal compounds such as glufosinate ammonium, bromoxynil,imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D).

Additional selectable markers can include phenotypic markers such asnucleic acid sequences encoding β-galactosidase, β-glucoronidase (GUS;Jefferson (1987) Plant Mol. Biol. Rep. 5:387-405); luciferase (Teeri etal. (1989) EMBO J. 8:343-350); anthocyanin production (Ludwig et al.(1990) Science 247:449-450), and fluorescent proteins such as greenfluorescent protein (GFP; Chalfie et al. (1994) Science 263:802-805;Fetter et al. (2004) Plant Cell 16:215-228; and Su et al. (2004)Biotechnol. Bioeng. 85:610-619); cyan fluorescent protein (CYP; Bolte etal. (2004) J. Cell Science 117:943-954; and Kato et al. (2002) PlantPhysiol. 129:913-942), and yellow fluorescent protein (PhiYFP™ availablefrom Evrogen (Moscow, Russia); Bolte et al. (2004) J. Cell Science117:943-954). For additional selectable markers, Baim et al. (1991)Proc. Natl. Acad. Sci. USA 88:5072-5076; Barkley & Bourgeois, “Repressorrecognition of operator and effectors” 177-120 In: The Operon (Miller &Reznikoff eds., Cold Spring Harbor Laboratory Press 1980); Bonin (1993)Ph.D. Thesis, University of Heidelberg; Brown et al. (1987) Cell49:603-612; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA89:6314-6318; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Deuschle et al. (1989) Proc. Natl. Acad. Sci. USA86:5400-5404; Deuschle et al. (1990) Science 248:480-483; Figge et al.(1988) Cell 52:713-722; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Gill et al. (1988) Nature 334:721-724; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen (1993) Ph.D.Thesis, University of Heidelberg; Hillenand-Wissman (1989) Topics Mol.Struc. Biol. 10:143-162; Hlavka et al., Handbook of ExperimentalPharmacology, Vol. 78 (Springer-Verlag 1985); Hu et al. (1987) Cell48:555-566; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Labowet al. (1990) Mol. Cell. Biol. 10:3343-3356; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Reines et al. (1993) Proc.Natl. Acad. Sci. USA 90:1917-1921; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Yao et al. (1992) Cell 71:63-72; Yarranton (1992) Curr.Opin. Biotech. 3:506-511; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; and Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956. The above list of selectable markers is not intended to belimiting, as any selectable marker can be used.

The vector therefore can be selected to allow introduction of theexpression cassette into the appropriate host cell such as a plant hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the cells are transfected with the plasmid vector DNA.

The present disclosure therefore includes nucleotide constructs such asexpression cassettes and vectors having a nucleotide sequence encoding amodified substrate protein of a pathogen-specific protease, where themodified substrate protein has a heterologous protease recognitionsequence. In addition, the nucleic acid constructs can include anucleotide sequence encoding a NB-LRR protein. The nucleic acidconstructs can be introduced into an organism such as a plant to conferresistance to plant pathogens expressing specific proteases.

Recombinant Peptides, Polypeptides and Proteins

Compositions of the present disclosure also include isolated orpurified, modified substrate proteins of a pathogen-specific protease,where the substrate proteins have heterologous protease recognitionsequences, as well as fragments and/or variants thereof. Methods forproducing peptide, polypeptides and proteins in plant cells, plant partsand plants are discussed elsewhere herein.

Methods of isolating or purifying peptides, polypeptides and proteinsare well known in the art. See, Ehle & Horn (1990) Bioseparation1:97-110; Hengen (1995) Trends Biochem Sci. 20:285-286; Basic Methods inProtein Purification and Analysis: A Laboratory Manual (Simpson et al.eds., Cold Spring Harbor Laboratory Press 2008); Regnier (1983) Science222:245-252; Shaw, “Peptide purification by reverse-phase HPLC” 257-287In: Methods in Molecular Biology, Vol. 32 (Walker ed., Humana Press1994); as well as US Patent Application Publication No. 2009/0239262;and U.S. Pat. Nos. 5,612,454; 7,083,948; 7,122,641; 7,220,356 and7,476,722.

As used herein, “peptide,” “polypeptide” and “protein” are usedinterchangeably to mean a polymer of amino acid residues. The termsapply to amino acid polymers in which one or more amino acid residues isan artificial chemical analogue of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.

As used herein, “residue,” “amino acid residue” and “amino acid” areused interchangeably to mean an amino acid that is incorporated intomolecule such as a peptide, polypeptide or protein. The amino acid canbe a naturally occurring amino acid and, unless otherwise limited, mayencompass known analogues of natural amino acids that can function in asimilar manner as naturally occurring amino acids.

As used herein, “recombinant,” when used in connection with a peptide,polypeptide or protein, means a molecule that has been created ormodified through deliberate human intervention such as by proteinengineering. For example, a recombinant polypeptide is one having anamino acid sequence that has been modified to include an artificialamino acid sequence or to include some other amino acid sequence that isnot present within its native/endogenous/non-recombinant form.

Further, a recombinant peptide, polypeptide or protein has a structurethat is not identical to that of any naturally occurring peptide,polypeptide or protein. As such, a recombinant peptide, polypeptide orprotein can be prepared by synthetic methods such as those known to oneof skill in the art.

If, and when, modified substrate proteins are to be isolated, completepurification is not required. For example, the modified substrateproteins described herein can be isolated and purified from normallyassociated material in conventional ways, such that in the purifiedpreparation, the proteins are the predominant species in thepreparation. At the very least, the degree of purification is such thatextraneous material in the preparation does not interfere with use ofthe proteins in the manner disclosed herein. The peptide, polypeptide orprotein can be at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98% or at least about 99% pure. Alternativelystated, the polypeptide is substantially free of cellular material suchthat preparations of the polypeptide can contain less than about 30%,25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (dry weight)of contaminating protein. When the polypeptide or an active variant orfragment thereof is recombinantly produced, culture medium representsless than about 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or 1% (dry weight) of chemical precursors or non-protein-of-interestchemicals.

It is known in the art that amino acids within the same conservativegroup can typically substitute for one another without substantiallyaffecting the function of a protein. For the purpose of the presentdisclosure, such conservative groups are set forth in Table 3 and arebased on shared properties. See also, Alberts et al., “Small molecules,energy, and biosynthesis” 56-57 In: Molecular Biology of the Cell(Garland Publishing Inc. 3^(rd) ed. 1994).

TABLE 3 Amino Acid Conservative Substitutions. Side Preferred Chain SideChain Hydropathy Conservative Residue Polarity pH Index Substitution Ala(A) Non-polar Neutral 1.8 Ser Arg (R) Polar Basic (strongly) −4.5 Lys,Gln Asn (N) Polar Neutral −3.5 Gln, His Asp (D) Polar Acidic −3.5 GluCys (C) Non-polar Neutral 2.5 Ser Gln (Q) Polar Neutral −3.5 Asn, LysGlu (E) Polar Acidic −3.5 Asp Gly (G) Non-polar Neutral −0.4 Pro His (H)Polar Basic (weakly) −3.2 Asn, Gln Ile (I) Non-polar Neutral 4.5 Leu,Val Leu (L) Non-polar Neutral 3.8 Ile, Val Lys (K) Polar Basic −3.9 Arg,Gln Met (M) Non-polar Neutral 1.9 Leu, Ile Phe (F) Non-polar Neutral 2.8Met, Leu, Tyr Pro (P) Non-polar Neutral −1.6 Gly Ser (S) Polar Neutral−0.8 Thr Thr (T) Polar Neutral −0.7 Ser Trp (W) Non-polar Neutral −0.9Tyr Tyr (Y) Polar Neutral −1.3 Trp, Phe Val (V) Non-polar Neutral 4.2Ile, Leu

The following six groups each contain amino acids that are typical butnot necessarily exclusive conservative substitutions for one another: 1.Alanine (A), Serine (S), Threonine (T); 2. Aspartic acid (D), Glutamicacid (E); 3. Asparagine (N), Glutamine (Q); 4. Arginine (R), Lysine (K);5. Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6.Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Substantial changes in function of a peptide, polypeptide or protein canbe made by selecting substitutions that are less conservative than thoselisted in the table above, that is, by selecting residues that differmore significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of substitution, (b) the charge orhydrophobicity of the polypeptide at the target site, or (c) the bulk ofa side chain. The substitutions that in general can be expected toproduce the greatest changes in the polypeptide's properties will bethose in which (a) a hydrophilic residue, for example, seryl orthreonyl, is substituted by a hydrophobic residue, for example, leucyl,isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline issubstituted by any other residue; (c) a residue having anelectropositive side chain, for example, lysyl, arginyl or histidyl, issubstituted by an electronegative side chain, for example, glutamyl oraspartyl; (d) a residue having a bulky side chain, for example,phenylalanyl, is substituted by a residue not having a side chain, forexample, glycyl; or (e) by increasing the number of sulfation orglycosylation.

In one aspect, the present disclosure is directed to an isolatedpolypeptide encoded by the recombinant nucleic acid molecule comprisingabout 90% identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8,wherein the polypeptide is a substrate protein of a plantpathogen-specific protease. In another embodiment, the isolatedpolypeptide can comprise about 95% identity to an amino acid sequenceselected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8,wherein the polypeptide is a substrate protein of a plantpathogen-specific protease. In other embodiments, the isolatedpolypeptide can comprise about 96% identity, about 97% identity, about98% identity and about 99% identity to an amino acid sequence selectedfrom SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, wherein thepolypeptide is a substrate protein of a plant pathogen-specificprotease.

An example of a modified substrate protein of a pathogen-specificprotease therefore includes SEQ ID NO:6 (modified PBS1 having an AvrRpt2protease recognition sequence). Another example of a modified substrateprotein of a pathogen-specific protease as described herein includes SEQID NO:8 (modified PBS1 having a TEV protease recognition sequence).Another example of a modified substrate protein of a pathogen-specificprotease includes PBS1 in which its endogenous AvrPphB cleavage site(SEQ ID NO:1) is replaced with a heterologous HopN1 cleavage site (SEQID NO:3). Another example of a modified substrate protein of apathogen-specific protease includes PBS1 in which its endogenous AvrPphBcleavage site (SEQ ID NO:1) is replaced with a heterologous SMV cleavagesite (SEQ ID NO:27). Another example of a modified substrate protein ofa pathogen-specific protease includes PBS1 in which its endogenousAvrPphB cleavage site (SEQ ID NO:1) is replaced with a heterologous BPMVcleavage site (SEQ ID NO:28). Another example of a modified substrateprotein of a pathogen-specific protease includes RIN4 in which itsendogenous AvrRpt2 cleavage site (SEQ ID NO:2) is replaced with aheterologous AvrPphB cleavage site (SEQ ID NO:1). Another example of amodified substrate protein of a pathogen-specific protease includes RIN4in which its endogenous AvrRpt2 cleavage site (SEQ ID NO:2) is replacedwith a heterologous TEV protease cleavage site (SEQ ID NO:4). Anotherexample of a modified substrate protein of a pathogen-specific proteaseincludes RIN4 in which its endogenous AvrRpt2 cleavage site (SEQ IDNO:2) is replaced with a heterologous HopN1 cleavage site (SEQ ID NO:3).Another example of a modified substrate protein of a pathogen-specificprotease includes RIN4 in which its endogenous AvrRpt2 cleavage site(SEQ ID NO:2) is replaced with a heterologous SMV cleavage site (SEQ IDNO:27). Another example of a modified substrate protein of apathogen-specific protease includes RIN4 in which its endogenous AvrRpt2cleavage site (SEQ ID NO:2) is replaced with a heterologous BPMVcleavage site (SEQ ID NO:28). As noted above, the endogenous proteasecleavage sequence, which is a preferred location for the heterologousprotease recognition sequence, typically can be located in an exposedloop of the substrate protein.

In addition to the full-length amino acid sequence of the modifiedsubstrate protein of the pathogen-specific protease, it is intended thatthe modified substrate protein can be a fragment or variant thereof thatis capable of being recognized by the plant pathogen protease and/or itscorresponding NB-LRR protein. For amino acid sequences, “fragment” meansa portion of the amino acid sequence of a reference polypeptide orprotein. Fragments of an amino acid sequence may retain the biologicalactivity of the reference polypeptide or protein. For example, less thanthe entire amino acid sequence of the modified substrate protein can beused and may have substrate protein activity and/or NB-LRR proteinbinding activity. Thus, fragments of the reference polypeptide orprotein can be at least about 150, 200, 250, 300, 350, 400 or 450 aminoacid residues, or up to the number of amino acid residues present in afull-length modified substrate protein. For example, about 80 aminoacids can be deleted from the N-terminus of PBS1 while retainingfunction. See, DeYoung et al. (2012), supra. Alternatively, about 100amino acids can be deleted from the C-terminus of PBS1 while retainingfunction. Id.

Likewise, a “variant” peptide, polypeptide or protein means asubstantially similar amino acid sequence to the amino acid sequence ofa reference peptide, polypeptide or protein. For amino acid sequences, avariant comprises an amino acid sequence derived from a referencepeptide, polypeptide or protein by deletion (so-called truncation) ofone or more amino acids at the N-terminal and/or C-terminal end of theamino acid sequence of the reference; deletion and/or addition of one ormore amino acids at one or more internal sites in the amino acidsequence of the reference; or substitution of one or more amino acids atone or more sites in the amino acid sequence of the reference. Variantpeptides, polypeptides or proteins encompassed by the present disclosureare biologically active, that is, they continue to possess the desiredbiological activity of the reference peptide, polypeptide or protein asdescribed herein. Such variants may result from, for example, geneticpolymorphism or human manipulation. Biologically active variants willhave at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to the amino acid sequenceof the reference peptide polypeptide or protein as determined bysequence alignment programs and parameters described above. For example,a biologically active variant of a modified substrate protein may differby as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, asfew as 5, as few as 4, 3, 2, or even 1 amino acid residue. It iscontemplated that PBS1 orthologues from other plant species can besubstituted for Arabidopsis PBS1, which typically have about 90% orhigher identity.

Deletions, insertions and substitutions of the modified substrateproteins are not expected to produce radical changes in thecharacteristics of the polypeptides. However, when it is difficult topredict the exact effect of the substitution, deletion or insertion inadvance of doing so, one of skill in the art will appreciate that theeffect can be evaluated by routine activity assays as described herein.

As above, variant peptides, polypeptides and proteins also encompasssequences derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more nucleic acid moleculescan be manipulated to encode new modified substrate proteins possessingthe desired properties. In this manner, libraries of recombinant nucleicacid molecules can be generated from a population of related nucleicacid molecules comprising sequence regions that have substantialsequence identity and can be homologously recombined in vitro or invivo. For example, using this approach, sequence motifs encoding adomain of interest can be shuffled between the nucleic acid moleculesidentified by the methods described herein and other known substrateprotein-encoding nucleic acid molecules to obtain a new nucleic acidmolecule that encodes a modified substrate protein with an improvedproperty such as increased activity or an expanded pH or temperaturerange. As such, a peptide, polypeptide or protein of the presentdisclosure can have many modifications.

The present disclosure therefore includes recombinant modified substrateproteins of pathogen-specific proteases, where the substrate proteinshave heterologous protease recognition sequences, as well as activefragments or variants thereof.

Transformed Plant Cells, Plant Parts and Plants

Compositions of the present disclosure also include transformed plantcells, plant parts and plants (i.e., subject plant cells, plant parts orplants) having a resistance to an increased number of plant pathogenswhen compared with control/native plant cells, plant parts or plants.

The transformed plant cells, plant parts or plants can have at least onenucleic acid molecule, nucleic acid construct, expression cassette orvector as described herein that encodes a modified substrate protein ofa pathogen-specific protease, where the modified substrate protein has aheterologous protease recognition sequence.

As used herein, “subject plant cell,” “subject plant part” or “subjectplant” means one in which a genetic alteration, such as transformation,has been effected as to a nucleic acid molecule of interest, or is aplant cell, plant part or plant that descended from a plant cell, plantpart or plant so altered and that comprises the alteration.

As used herein, “control plant cell,” “control plant part” or “controlplant” means a reference point for measuring changes in phenotype of thesubject plant cell, plant part or plant. A control plant cell, plantpart or plant can comprise, for example: (a) a wild-type plant cell,plant part or plant (i.e., of the same genotype as the starting materialfor the genetic alteration that resulted in the subject plant cell,plant part or plant); (b) a plant cell, plant part or plant of the samegenotype as the starting material, but which has been transformed with anull construct (i.e., with a construct that has no known effect on thetrait of interest, such as a construct comprising a marker gene); (c) aplant cell, plant part or plant that is a non-transformed segregantamong progeny of a subject plant cell, plant part or plant; (d) a plantcell, plant part or plant genetically identical to the subject plantcell, plant part or plant, but which is not exposed to conditions orstimuli that would induce expression of the gene of interest; or (e) thesubject plant cell, plant part or plant itself, under conditions inwhich the nucleic acid molecule/construct of interest is not expressed.

Methods of introducing nucleotide sequences into plants, plant parts orplant host cells are well known in the art and are discussed in greaterdetail below.

As used herein, “plant cell” or “plant cells” means a cell obtained fromor found in seeds, suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollenand microspores. Plant cell also includes modified cells, such asprotoplasts, obtained from the aforementioned tissues, as well as plantcell tissue cultures from which plants can be regenerated, plant calliand plant clumps.

As used herein, “plant part” or “plant parts” means organs such asembryos, pollen, ovules, seeds, flowers, kernels, ears, cobs, leaves,husks, stalks, stems, roots, root tips, anthers, silk and the like.

As used herein, “plant” or “plants” means whole plants and theirprogeny. Progeny, variants and mutants of the regenerated plants alsoare included, provided that they comprise the introduced nucleic acidmolecule.

As used herein, “grain” means mature seed produced by commercial growersfor purposes other than growing or reproducing the species. The class ofplants that can be used in the methods described herein is generally asbroad as the class of higher plants amenable to transformationtechniques, including both monocotyledonous (monocots) anddicotyledonous (dicots) plants.

Examples of plant species of interest herein include, but are notlimited to, corn (Zea mays), Brassica spp. (e.g., B. napus, B. rapa, B.juncea), particularly those Brassica species useful as sources of seedoil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats (Avena sativa), barley (Hordeumvulgare), vegetables, ornamentals, and conifers.

Vegetables of interest include, but are not limited to, tomatoes(Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans(Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrusspp.), and members of the genus Cucumis such as cucumber (C. sativus),cantaloupe (C. cantalupensis), and musk melon (C. melo).

Ornamentals of interest include, but are not limited to, azalea(Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus(Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.),daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), andchrysanthemum.

Conifers of interest include, but are not limited to, pines such asloblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine(Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine(Pinus radiata); Douglas fir (Pseudotsuga menziesii); Western hemlock(Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoiasempervirens); true firs such as silver fir (Abies amabilis) and balsamfir (Abies balsamea); and cedars such as Western red cedar (Thujaplicata) and Alaska yellow cedar (Chamaecyparis nootkatensis).

In some instances, the plant cells, plant parts or plants of interestare crop plants (e.g., corn, alfalfa, sunflower, Brassica, soybean,cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.).

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

The present disclosure therefore includes transgenic plant cells, plantparts and plants having incorporated therein at least one nucleic acidmolecule that encodes a modified substrate protein of apathogen-specific protease, where the modified substrate protein has aheterologous protease sequence, to confer disease resistance to plantpathogens expressing specific proteases.

Methods

Methods of the present disclosure include introducing and expressing ina plant cell, plant part or plant a nucleic acid molecule or constructas described herein. As used herein, “introducing” means presenting tothe plant cell, plant part or plant, a nucleic acid molecule orconstruct in such a manner that it gains access to the interior of acell of the plant. The methods do not depend on the particular methodfor introducing the nucleic acid molecule or nucleic acid construct intothe plant cell, plant part or plant, only that it gains access to theinterior of at least one cell of the plant or plant part. Methods ofintroducing nucleotide sequences, selecting transformants andregenerating whole plants, which may require routine modification inrespect of a particular plant species, are well known in the art. Themethods include, but are not limited to, stable transformation methods,transient transformation methods, virus-mediated methods and sexualbreeding. As such, the nucleic acid molecule or construct can be carriedepisomally or integrated into the genome of the host cell.

As used herein, “stable transformation” means that the nucleic acidmolecule or construct of interest introduced into the plant integratesinto the genome of the plant and is capable of being inherited by theprogeny thereof. As used herein, “transient transformation” means thatthe nucleic acid molecule or construct of interest introduced into theplant is not inherited by progeny.

Methods of transforming plants and introducing a nucleotide sequence ofinterest into plants can and will vary depending on the type of plant,plant part or plant host cell (i.e., monocotyledonous or dicotyledonous)targeted for transformation. Methods of introducing nucleotide sequencesinto plant host cells therefore include Agrobacterium-mediatedtransformation (e.g., A. rhizogenes or A. tumefaciens; U.S. Pat. Nos.5,563,055 and 5,981,840), calcium chloride, direct gene transfer(Paszkowski et al. (1984) EMBO J. 3:2717-2722), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), microinjection(Crossway et al. (1986) Biotechniques 4:320-334), microprojectilebombardment/particle acceleration (McCabe et al. (1988) Biotechnology6:923-926; and Tomes et al., “Direct DNA transfer into intact plantcells via microprojectile bombardment” In: Plant Cell, Tissue, and OrganCulture: Fundamental Methods (Gamborg & Phillips eds., Springer-Verlag1995); as well as U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and5,932,782), polyethylene glycol (PEG), phage infection, viral infection,and other methods known in the art. See also, EP Patent Nos. 0 295 959and 0 138 341.

A nucleic acid molecule or construct as described above herein can beintroduced into the plant cell, plant part or plant using a variety oftransient transformation methods. Methods of transiently transformingplant cells, plant parts or plants include, but are not limited to,Agrobacterium infection, microinjection or particle bombardment. See,Crossway et al. (1986) Mol. Gen. Genet. 202:179-185; Hepler et al.(1994) Proc. Natl. Acad. Sci. USA 91:2176-2180; Hush et al. (1994) J.Cell Sci. 107:775-784; and Nomura et al. (1986) Plant Sci. 44:53-58.Alternatively, the plant cell, plant part or plant can be transformed byviral vector systems or by precipitation of the nucleic acid molecule orconstruct in a manner that precludes subsequent release of the DNA.Thus, transcription from the particle-bound nucleotide sequence canoccur, but the frequency with which it is released to become integratedinto the genome is greatly reduced. Such methods include the use ofparticles coated with polyethylimine (PEI; Sigma; St. Louis, Mo.).

Likewise, the nucleic acid molecules or constructs as described hereincan be introduced into the plant cell, plant part or plant by contactingit with a virus or viral nucleic acids. Generally, such methods involveincorporating the nucleic acid molecule or construct within a viral DNAor RNA molecule. It is recognized that the nucleotide sequences can beinitially synthesized as part of a viral polyprotein, which later can beprocessed by proteolysis in vivo or in vitro to produce the desiredrecombinant protein. Methods for introducing nucleotide sequences intoplants and expressing the protein encoded therein, involving viral DNAor RNA molecules, are well known in the art. See, Porta et al. (1996)Mol. Biotechnol. 5:209-221; as well as U.S. Pat. Nos. 5,866,785;5,889,190; 5,889,191 and 5,589,367.

Methods also are known in the art for the targeted insertion of anucleic acid molecule or construct at a specific location in the plantgenome. In some instances, insertion of the nucleic acid molecule orconstruct at a desired genomic location can be achieved by using asite-specific recombination system. See, Int'l Patent ApplicationPublication Nos. WO 99/025821, WO 99/025854, WO 99/025840, WO 99/025855and WO 99/025853.

Transformation techniques for monocots therefore are well known in theart and include direct gene uptake of exogenous nucleic acid moleculesor constructs by protoplasts or cells (e.g., by PEG- orelectroporation-mediated uptake, and particle bombardment into callustissue). Transformation of monocots via Agrobacterium also has beendescribed. See, Int'l Patent Application Publication No. WO 94/00977 andU.S. Pat. No. 5,591,616; see also, Christou et al. (1991) Bio/Technology9:957-962; Datta et al. (1990) Bio/Technology 8:736-740; Fromm et al.(1990) Biotechnology 8:833-844; Gordon-Kamm et al. (1990) Plant Cell2:603-618; Koziel et al. (1993) Bio/Technology 11:194-200; Murashige &Skoog (1962) Physiologia Plantarum 15:473-497; Shimamoto et al. (1989)Nature 338:274-276; Vasil et al. (1992) Bio/Technology 10:667-674; Vasilet al. (1993) Bio/Technology 11:1553-1558; Weeks et al. (1993) PlantPhysiol. 102:1077-1084; and Zhang et al. (1988) Plant Cell Rep.7:379-384; as well as EP Patent Application Nos. 0 292 435; 0 332 581and 0 392 225; Int'l Patent Application Publication Nos. WO 93/07278 andWO 93/21335; and U.S. Pat. No. 7,102,057.

Transformation techniques for dicots also are well known in the art andinclude Agrobacterium-mediated techniques and techniques that do notrequire Agrobacterium. Non-Agrobacterium-mediated techniques include thedirect uptake of exogenous nucleic acid molecules by protoplasts orcells (e.g., by PEG- or electroporation-mediated uptake, particlebombardment, or microinjection). See, Klein et al. (1987) Nature327:70-73; Paszkowski et al. (1984) EMBO J. 3:2717-2722; Potrykus et al.(1985) Mol. Gen. Genet. 199:169-177; and Reich et al. (1986)Bio/Technology 4:1001-10041; as well as U.S. Pat. No. 7,102,057.

Plant cells that have been transformed can be grown into plants bymethods well known in the art. See, McCormick et al. (1986) Plant CellRep. 5:81-84. These plants then can be grown, and either pollinated withthe same transformed strain or different strains, and the resultingprogeny having the desired phenotypic characteristic identified. Two ormore generations can be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited, and thenseeds harvested to ensure expression of the desired phenotypiccharacteristic has been achieved.

The present disclosure therefore provides methods of introducing intoplants, plant parts and plant host cells the nucleic acid constructsdescribed herein, for example, an expression cassette of the presentdisclosure, which encode a modified substrate protein of apathogen-specific protease, where the substrate protein has aheterologous protease recognition sequence.

EXAMPLES

The disclosure will be more fully understood upon consideration of thefollowing non-limiting examples, which are offered for purposes ofillustration, not limitation.

Example 1

RPS5 Activation by AvrRpt2 when Transiently Co-Expressed with a ModifiedPBS1 Protein Containing an AvrRpt2 Cleavage Site.

Methods:

Transient Transformation:

PBS1^(RCS2) (SEQ ID NO: 4; modified PBS1 containing an AvrRpt2 cleavagesite) was inserted in a vector (pTA7002; Aoyama & Chua (1997), supra)containing a dexamethasone-inducible promoter as described in DeYoung etal. (2012), supra. This vector was transformed into A. tumfaciens strainGV3101(pMP90). RPS5 and AvrRpt2 genes also were inserted into pTA7002(separate constructs) as described in DeYoung et al. (2012) andindependently transformed into GV3101(pMP90). For use as controls,GV3101(pMP90) also was transformed with the empty vector pTA7002, andwith pTA7002 containing the wild-type PBS1 gene, and with pTA7002containing AvrPphB, creating a total of six strains (listed below).

To transiently express these genes in plants, the Agrobacterium strainswere prepared as described in DeYoung et al. (2012), supra, mixed inequal ratios in the combinations shown in FIGS. 1A and 1B and injectedinto expanding leaves of 4-week-old N. glutinosa plants. Proteinexpression was induced by spraying leaves with 50 μM dexamethasone 40hours after injection.

For evaluation of cell death, leaves were scored for visible collapse 24hours after dexamethasone application. Electrolyte leakage (aquantitative indicator of cell death) was measured as described inDeYoung et al. (2012), supra.

Constructs (all in pTA7002):

-   -   1. Wild-type PBS1;    -   2. PBS1 with the AvrRpt2 Cleavage Site (PBS1^(RCS2));    -   3. AvrRpt2;    -   4. AvrPphB;    -   5. RPS5; and    -   6. Empty Vector (pTA7002).

Results: As shown in FIG. 1, strong leaf collapse was induced whenwild-type PBS1 was co-expressed with RPS5 and AvrPphB (far right side offigure). This is the positive control and demonstrated that ArabidopsisRPS5 can induce defense responses (cell death) when activated in N.glutinosa. Co-expression of wild-type PBS1 and RPS5 with AvrRpt2 did notinduce significant collapse (left most leaf, right half), consistentwith the inability of AvrRpt2 to cleave wild-type PBS1.

In contrast, co-expression of PBS1^(RCS2) with RPS5 and AvrRpt2 inducedstrong leaf collapse (left side of leaves 1 and 2), demonstrating thatRPS5 can be activated by PBS1^(RCS2) cleavage. FIG. 2 quantifies thelevel of cell death in each treatment, and is consistent with the visualsymptoms shown in FIG. 1A.

This example therefore shows that the AvrPphB cleavage site within thePBS1 activation loop can be replaced with the recognition sequence for adifferent protease from P. syringae named AvrRpt2. This replacementmakes PBS1 a substrate protein for AvrRpt2 instead of AvrPphB. As such,co-expression of the modified PBS1 with AvrRpt2 and RPS5 resulted inactivation of RPS5, whereas co-expression of wild-type PBS1 with AvrRpt2and RPS5 did not.

Example 2

Transformation of A. thaliana with Modified PBS1 Protein Containing anAvrRpt2 Cleavage Site Confers Resistance to P. syringae StrainsExpressing AvrRpt2.

Methods:

Stable Transformation:

An A. thaliana mutant line containing mutations in the RPS2 and RIN4genes (makes A. thaliana susceptible to infection by P. syringaeexpressing AvrRpt2) was stably transformed with a PBS1^(RCS2) constructusing A. tumefaciens strain GV3101(pMP90) following the protocol ofClough & Bent using resistance to the herbicide glufosinate as aselectable marker. See, Clough & Bent (1998) Plant J. 16:735-743. Fiveindependent transgenic plants were selected. Leaves of these individualplants were inoculated with P. syringae strain DC3000 expressing AvrRpt2at a concentration of 0.5×10⁷ colony forming units (cfu) per milliliterusing a needleless 1 mL syringe. Leaves were scored for visible leafcollapse (cell death) 24 hours after injection. Wild-type A. thalianawas used as a positive control.

Results:

As shown in FIG. 3, transgenic Hines 1, 2, 4 and 5 showed leaf collapseon the right side in response to inoculation with P. syringae strainDC3000(AvrRpt2), which is indicative of programmed cell death activatedby RPS5 in response to AvrRpt2. Line 3 did not show leaf collapse,likely due to failure of the PBS1^(RCS2) transgene to express. This lackof leaf collapse demonstrates that the parent rin4rps2 mutant line doesnot induce cell death at this time point in response to inoculation withDC3000(AvrRpt2), which has been reported previously by Day et al. See,Day et al. (2005) Plant Cell 17:1292-1305. Activation of cell deathindicates that transgenic lines 1, 2, 4 and 5 have gained diseaseresistance to DC3000(AvrRpt2); thus, expression of PBS1^(RCS2) enablesthe endogenous RPS5 gene of Arabidopsis to confer resistance to thisstrain.

Example 3 (Prophetic)

RPS5 Activation by a Protease (BEC1019) from a Powdery Mildew Funguswhen Co-Expressed with a Modified PBS1 Protein Containing a BEC1019Cleavage Site.

The genome sequences of several different species of powdery mildewfungi, including species that infect wheat and barley (Blumeriagraminis) and species that infect Arabidopsis (Golovinomycescichoracearum and G. orontii) have been determined. These genomes havebeen analyzed for the presence of protease enzymes that are likelysecreted during infection of host plants. One such protease that isconserved among these fungal species has been identified and has beennamed BEC1019. Silencing of the BEC1019 gene has been shown tocompromise virulence of barley powdery mildew, indicating that thisprotease is required to cause disease, at least on barley ((Pliego, C.,Nowara, D., Bonciani, G., Gheorghe, D. M., Xu, R., Surana, P., Whigham,E., Nettleton, D., Bogdanove, A. J., Wise, R. P., Schweizer, P.,Bindschedler, L. V., and Spanu, P. D. 2013. Host-induced gene silencingin barley powdery mildew reveals a class of ribonuclease-like effectors.Mol Plant Microbe Interact 26:633-642).

A nucleic acid molecule for the protease recognition sequence forBEC1019 will be inserted into the activation loop of PBS1. The modifiedPBS1 nucleic acid molecule then will be transformed into Arabidopsisplants lacking a functional PBS1 gene, but that are wild-type for RPS5.The Arabidopsis plants should become resistant to infection by powderymildew species such as G. cichoracearum and G. golovinomyces. If this isconfirmed, RPS5 and PBS1 containing the BEC1019 cleavage site will betransformed into various crop plants to confer resistance to powderymildew (e.g., wheat, barley, grapevine, etc.).

Example 4 (Prophetic)

RPS5 Activation by Tobacco Etch Virus (TEV) Protease when Co-Expressedwith a Modified PBS1 Protein Containing a TEV Polyprotein Cleavage Site.

Several viruses that infect plants encode proteases that are requiredfor processing of viral polyproteins. A nucleic acid molecule encodingthe protease recognition sequence for TEV protease will be inserted intothe activation loop of PBS1. The modified PBS1 and wild-type RPS5nucleic acid molecules will be used to transform the tobacco relativeNicotiana benthamiana. Activation of RPS5 by TEV protease will first betested using the transient expression system described in Example 1.Assuming that RPS5 is activated, as predicted, the modified PBS1 geneand RPS5 will be transformed into N. tabacum and the resultingtransgenic plants are tested for resistance to TEV infection. This genepair should confer resistance.

Example 5

RPS5 Activation by AvrRpt2 when Transiently Co-Expressed with a ModifiedPBS1 Protein Containing an AvrRpt2 Cleavage Site.

Methods:

Construction of plasmids for transgene expression. A PBS1::RCS2 entryclone harboring the RIN4 cleavage site sequence inserted at the AvrPphBcleavage site was constructed using overlap PCR and a pBSDONR PBS1template. A Multisite Gateway LR Clonase reaction was performed torecombine the entry clone, the pBAV154 destination vector (carrying adexamethasone-inducible promoter) and a clone containing a 3×HAC-terminal epitope. The PBS1^(RCS2) and PBS1^(TCS) entry clones in whichthe AvrPphB cleavage site of PBS1 was replaced with the RIN4 cleavagesite 2 (RCS2) and TEV cleavage site (TCS), respectively, were createdfrom the pBSDONR PBS1 template using site-directed mutagenesis PCR.These entry clones and 3×HA were recombined into the pTA7002 destinationvector (carrying a dexamethasone-inducible promoter) using LR reactions.The coding regions of AvrPphB, AvrRpt2, C122A (AvrRpt2 mutant), and TEVprotease were PCR-amplified and cloned into the Gateway vector pBSDONRP1-P4 using Gateway BP Clonase to generate entry clones, which wererecombined with pTA7002 and 5×Myc using LR reactions. To generate plantexpression constructs for PBS1^(RCS2) fused to 3×HA driven by the nativePBS1 regulatory elements (pPBS1-PBS1^(RCS)-HA), an 875 bp Apal/Xholfragment spanning the PBS1 promoter and a 400 bp Notl/Sacl fragmentspanning the PBS1 terminator, and a 1731 bp Xhol/Xbal fragmentcontaining the Gateway cassette were inserted into the pGreen0229 binaryvector. PBS1^(RCS2) and 3×HA were then recombined into this destinationconstruct using LR clonase. All constructs were verified by sequencing.Primer sequences used in cloning are listed in Table 4 below.

TABLE 4 Primer Sequences. Primer Name Sequence (5′→3′) (SEQ ID NO:)Purpose SHP5 GTGCCTAAATTCGGTGACTGGTCTCATGTCTCCACTAG PBS1::RCS2AGT (SEQ ID NO: 11) SHP6 CCAGTCACCGAATTTAGGCACTTTGTCTCCCGTTGGTCPBS1::RCS2 C (SEQ ID NO: 12) SHP28GTGCCTAAATTCGGTGACTGGACTAGAGTTATGGGAAC PBS1^(RCS2)TTATGGT (SEQ ID NO: 13) SHP29 CCAGTCACCGAATTTAGGCACCGTTGGTCCGAGTTTAGPBS1^(RCS2) CAA (SEQ ID NO: 14) SHP59GAAAACCTGTATTTTCAGGGCACTAGAGTTATGGGAACT PBS1^(TCS)TATGGT (SEQ ID NO: 15) SHP60 GCCCTGAAAATACAGGTTTTCCGTTGGTCCGAGTTTAGPBS1^(TCS) CAA (SEQ ID NO: 16) SHP61GGACAAGTTTGTACAAAAAAGCAGGCTCTATGGAAAGC TEV proteaseTTGTTTAAGGGG (SEQ ID NO: 17) SHP62GGACAACTTTGTATAGAAAAGTTGGGTGATTCATGAGTT TEV proteaseGAGTCGCTTC (SEQ ID NO: 18) SHP15 GGACAAGTTTGTACAAAAAAGCAGGCTCTATGAAAATTGAvrRpt2 or CTCCAGTTGCCA (SEQ ID NO: 19) C122A SHP16GGACAACTTTGTATAGAAAAGTTGGGTGGCGGTAGAGC AvrRpt2 orATTGCGTGTGG (SEQ ID NO: 20) C122A RB63GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGGGT AvrPphBGTGCATCCTCTTCAGG (SEQ ID NO: 21) RB62GGGGACAACTTTGTATAGAAAAGTTGGGTGCGAAACTC AvrPphBTAAACTCGTTTA (SEQ ID NO: 22) BD113 AGGGCCCATAGTTTCGTTCTCTGCTTCAA (SEQ IDPBS1 promoter NO: 23) BD115 ACTCGAGCTCCTCCTTTACTCAATTTTC (SEQ ID NO: 24)PBS1 promoter BD131 AGCGGCCGCAACCGGTTTGGGTCGGTCTTG (SEQ ID PBS1 NO: 25)terminator BD119 AGAGCTCCATGTGACCCACGTTGTCCGA (SEQ ID PBS1 NO: 26)terminator

Plant materials and growth conditions. Arabidopsis thaliana, Nicotinabenthamiana, and N. glutinosa plants were grown under a 9 h light/15 hdark cycle at 24° C. in Metro-Mix 360 plotting mixture (Sun GroHorticulture, http://www.sungrow.com). Transfer-DNA insertion lines ofPBS1 (pbs1-7; Salk_062464C) and RPS5 (rps5-3; Salk_015294C) wereobtained from the Salk T-DNA Express collection via the ArabidopsisBiological Resource Center at Ohio State University.

Assessing resistance to bacterial infection in Arabidopsis. Forhypersensitive response (HR) assays, Pseudomonas syringae strainsDC3000(avrPphB) and DC3000(avrRpt2) were grown on King's medium B agarplates, and infiltrated into 5-week old Arabidopsis leaves usingneedless syringe at 10⁸ colony-forming units (cfu) per ml (OD₆₀₀=0.1).Leaves were scored and photographed 21 hours after inoculation. Tomeasure bacterial growth within plant leaves, 5-week-old Arabidopsisplants were infiltrated with DC3000(avrRpt2) at 10⁵ cfu per ml. A totalof 0.5 cm² of leaf tissue, collected using a cork borer, was ground in10 mM MgCl₂ and plated by serial dilution on selective medium (King'smedium B supplemented with 100 μg/mL rifampicin and 50 μg/mL kanamycin)in four replicates at the indicated time points.

Transient expression assays in Nicotiana species. For transientexpression assays, the dexamethasone-inducible constructs describedabove were mobilized into Agrobacterium tumefaciens strain GV3101. Afterovernight culture in liquid LB media, bacterial cells were pelleted andresuspended in 10 mM MgCl₂ with 100 μM acetosyringone (Sigma-Aldrich),adjusted to an OD₆₀₀ of 0.1, incubated for 2 hours at room temperatureand infiltrated into leaves of 4-week-old N. benthamiana or N. glutinosaplants. Leaves were sprayed with 50 μM dexamethasone 40 hours afterinjection. Samples were harvested for protein extraction 6 or 24 hoursafter dexamethasone application, and HR was evaluated 24 hours afterdexamethasone application.

Electrolyte leakage assays. To measure electrolyte leakage fromAgrobacterium-infiltrated Nicotiana leaves, 8 leaf discs (6 mm indiameter) were collected from four individual leaves at 2 hours postdexamethasone induction. After washing three times with distilled water,the leaf discs were floated in 5 ml of distilled water containing 0.001%Tween 20 (Sigma-Aldrich). Conductivity was monitored in four replicatesat the indicated time points using a Traceable Pen Conductivity Meter(VWR).

Immunoblot analysis. Nicotiana leaf tissue expressing a protein ofinterest was ground in extraction buffer (150 mM NaCl, 50 mM Tris [pH7.5], 0.2% Nonidet P-40 [Sigma-Aldrich], 1% plant protease inhibitorcocktail [Sigma-Aldrich]). Cell debris was pelleted at 12,000 rpm for 10min., and the collected supernatants were separated on a 4-20% gradientTris-Hepes-SDS polyacrylamide gel (Thermo Scientific). Proteins weredetected with 1:2000 diluted peroxidase-conjugated anti-HA antibody(Sigma-Aldrich) or with 1:4000 diluted peroxidase-conjugated anti-c-Mycantibody (Roche). Total protein from transgenic Arabidopsis tissueexpressing pDEX-PBS1::RCS2-HA was prepared 16 hours post dexamethasoneinduction, and subjected to immunoblot analysis. Total protein fromtransgenic Arabidopsis tissue expressing pPBS1-PBS1^(RCS2)-HA wasprepared from healthy plants, or 12 hours post inoculation withDC3000(e.v.) or DC3000(avrRpt2) at a density of 10⁸ cfu/ml, andimmunoprecipitated using Pierce Anti-HA agarose (Thermo Scientific) forimmunoblot analysis.

Results: As illustrated in FIG. 4, seven amino acids flanking theAvrPphB cleavage site in PBS1 (GDKSHVS; SEQ ID NO:1) were replaced withthe RIN4 cleavage site 2 (RCS2) sequence (VPKFGDW; SEQ ID NO:2).Co-expression of the PBS1^(RCS2) with AvrRpt2 and PRS5 induced anRPS5-dependent cell death response, whereas cell death was not detectedin the absence of AvrRpt2 or PBS1^(RCS2) (FIG. 5). A protease-deficientmutant form of AvrRpt2 (C122A) induced only a very weak macroscopicresponse. To quantify the HR, electrolyte leakage analysis was performedas a measurement of cell death. Consistent with the macroscopicsymptoms, PBS1^(RCS2) with AvrRpt2 induced as much electrolyte leakageas wild-type PBS1 cleaved by AvrPphB, whereas PBS1^(RCS2) with C122Aonly weakly activated RPS5 (FIG. 6). Immunoblot analysis confirmed thatAvrRpt2 cleaved PBS1^(RCS2) at 6 hours post-induction, whereas C122A orAvrPphB did not (FIG. 7). At 24 hours post-induction, AvrRpt2-inducedcleavage was increased, and even C122A induced small amounts of cleavage(FIG. 7), consistent with the observed weak induction of cell death bythis construct (FIGS. 5 and 6). Together, these data establish thatPBS1^(RCS2) is a substrate for AvrRpt2 and that AvrRpt2-mediatedcleavage activates RPS5.

To assess whether AvrRpt2-mediated cleavage of PBS1^(RCS2) can activateRPS5 expressed at native levels in Arabidopsis, an Arabidopsis rin4rps2mutant was stably transformed with PBS1^(RCS2) under the native PBS1regulatory elements (pPBS1-PBS1^(RCS2)-HA/rin4rps2). The rin4rps2 mutantwas used to avoid activation of the endogenous RPS2 disease resistanceprotein by AvrRpt2. As shown in FIG. 8, two independent transgenic lines(#5 and #2) showed a visible HR 21 hours after inoculation with P.syringae strain DC3000(avrRpt2), whereas the untransformed rin4rps2mutant did not. In planta bacterial growth assays showed that growth ofDC3000(avrRpt2) in transgenic lines #5 and #2 was restricted to levels100- to 200-fold less compared to rin4rps2, while transgenic lines #1and #3 had approximately 5-10 fold lower bacterial growth than rin4rps2(FIG. 9; statistically significant differences were determined by atwo-tailed Student's t-Test (P<0.01) or a one-way ANOVA and Tukey's HSD(P<0.01)). Restriction of bacterial growth correlated with expressionlevels of PBS1^(RCS2) (FIG. 10). Proteins from transgenic lines shown inFIG. 8 were immunoprecipitated with anti-HA agarose, and immunoblotswere performed with an anti-HA antibody. In addition, a cleavage productof PBS1^(RCS2) was detected in transgenic line #5 twelve hours afterinoculation with DC3000(avrRpt2), but not with DC3000 lacking avrRpt2(DC3000(e.v.); FIG. 11), indicating that cleavage of PBS1^(RCS2) byAvrRpt2 activates RPS5 in Arabidopsis. In addition, these transgenicplants also displayed HR 21 hours after injection with DC3000(avrPphB),demonstrating that native recognition specificity of RPS5 was retainedin these transgenic lines (FIG. 12). Thus, RPS5-mediated diseaseresistance can be activated by two different protease effector proteinsin the PBS1^(RCS2) transgenic plants, demonstrating that the recognitionspecificity of RPS5 can be expanded by addition of new ‘decoy’ copies ofPBS1.

To test whether this decoy approach could be extended to recognizepathogens beyond P. syringae, a PBS1 decoy was created that can becleaved by the NIa protease of Tobacco Etch Virus (referred to asPBS1^(TCS)) (FIG. 13). TEV is a positive stranded RNA virus that encodesa polyprotein that must be post-translationally processed by itsembedded Nla protease. This protease is essential for viral replication,thus an R protein that is triggered by its enzymatic activity should behighly durable, as it would be extremely difficult for the virus tosimultaneously change the specificity of its protease and the proteasecleavage sites embedded within its polyprotein.

TEV protease and RPS5 were transiently co-expressed with PBS1^(TCS) inN. benthamiana (FIG. 14). RPS5-mediated cell death was induced only whenRPS5 was co-expressed with PBS1^(TCS) and TEV protease, but was notinduced when either PBS1^(TCS) or TEV protease was excluded (FIG. 15).Quantification of cell death using electrolyte leakage showed thatPBS1^(TCS) and TEV protease induced RPS5-mediated cell death equivalentto wild-type PBS1 and AvrPphB (FIG. 16). Immunoblot analysis confirmedthat TEV protease cleaved PBS1^(TCS) 6 hours post induction, whereasAvrPphB did not. Also, TEV protease did not cleave wild-type PBS1. Thesedata established that PBS1 can be engineered to function as a decoy todetect the presence of proteases from two very different classes ofpathogen, viruses and bacteria, and open the way to engineeringresistance to a broad array of pathogens.

Example 6 Recognition of AvrPphB by Soybean.

In this Example, P. syringae pv. glycinea Race4 strains carrying AvrPphBor AvrB::Ω (a non-functional effector used as an empty vector control)were infiltrated into a unifoliolate leaf of soybean cultivar Flambeau.The leaf was removed from the plant 24 hours after injection, clearedusing hot 70% ethanol and photographed.

Soybean responded to P. syringae expressing AvrPphB with an HR asindicated by leaf browning (dark region shown in FIG. 17). These dataindicated that using the decoy approach described herein to engineerresistance in crop plants might not require transfer of the ArabidopsisRPS5 gene if crop plants already possess the ability to detect AvrPphBby a similar mechanism. In addition, PBS1 is highly conserved among cropplants, including soybean. It may thus be possible to engineer soybean,and other crop plants, to detect various pathogen proteases by makingsmall changes to their endogenous PBS1 genes.

All of the patents, patent applications, patent application publicationsand other publications recited herein are hereby incorporated byreference as if set forth in their entirety.

The present disclosure has been described in connection with what arepresently considered to be the most practical and preferred embodiments.However, the present disclosure has been presented by way ofillustration and is not intended to be limited to the disclosedembodiments. Accordingly, one of skill in the art will realize that thepresent disclosure is intended to encompass all modifications andalternative arrangements of the compositions and methods as set forth inthe appended claims.

1. A recombinant nucleic acid molecule comprising a heterologouspromoter operably linked to a nucleotide sequence that encodes at leastone substrate protein of a plant pathogen-specific protease expressed bythe plant pathogen having a heterologous pathogen-specific proteaserecognition sequence, wherein the substrate protein is Arabidopsisthaliana Resistance To Pseudomonas syringae pv. maculicola 1 (RPM1)Interacting Protein 4 (RIN4), and wherein the endogenous AvrRpt2cleavage site of SEQ ID NO:2 is replaced with a heterologouspathogen-specific protease recognition sequence selected from the groupconsisting of GDKSHVS (SEQ ID NO:1), QEHGCQL (SEQ ID NO:3), ENLYFQG (SEQID NO:4), EPVSTQG (SEQ ID NO:27) and PVVQAQS (SEQ ID NO:28). 2.(canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled)
 6. (canceled) 7.The recombinant nucleic acid molecule of claim 1, wherein theheterologous pathogen-specific protease recognition sequence is locatedbetween about amino acid position 142 to about amino acid position 164when the substrate protein is RIN4.
 8. A modified substrate protein of aplant pathogen-specific protease expressed by the plant pathogencomprising an amino acid sequence having a heterologous proteaserecognition sequence, wherein the modified substrate protein is encodedby the recombinant nucleic acid molecule according to claim
 1. 9. Avector comprising the recombinant nucleic acid molecule according toclaim
 1. 10. A transformed plant cell comprising the recombinant nucleicacid molecule according to claim
 1. 11. The transformed plant cell ofclaim 10, wherein the plant cell is from a plant selected from the groupconsisting of a monocot and a dicot.
 12. A transformed plant comprisingthe recombinant nucleic acid molecule according to claim
 1. 13. Thetransformed plant of claim 12, wherein the plant is selected from thegroup consisting of a monocot and a dicot.
 14. A transgenic seed of thetransformed plant according to claim
 12. 15. A method of protecting aplant from infection by a plant pathogen that secretes at least onespecific protease, the method comprising the step of: introducing to theplant a nucleotide sequence that encodes at least one substrate proteinof a plant pathogen-specific protease secreted by the plant pathogenhaving a heterologous pathogen-specific protease recognition sequencewithin the substrate protein, wherein the substrate protein isArabidopsis thaliana Resistance To Pseudomonas syringae pv. maculicola 1(RPM1) Interacting Protein 4 (RIN4), and wherein the endogenous AvrRpt2cleavage site of SEQ ID NO:2 is replaced with a heterologouspathogen-specific protease recognition sequence selected from the groupconsisting of GDKSHVS (SEQ ID NO:1), QEHGCQL (SEQ ID NO:3), ENLYFQG (SEQID NO:4), EPVSTQG (SEQ ID NO:27) and PVVQAQS (SEQ ID NO:28). 16.(canceled)
 17. (canceled)
 18. (canceled)
 19. (canceled)
 20. (canceled)